Class-12

CLASS ‐ XII S. No. Topic Page No.

1. Syllabus Guide Line 04

2. Biotechnology - an Introduction 13

3. Biotechnology Through The Ages 14

4. Biotechnology Development in India 20

5. Nanobiotechnology 24

6. Bioinformatics 30

7. Incredible Edible Vaccines 34

8. Discovery of DNA Computer 36

9. Gene Therapy 41

10. DNA Fingerprinting Analysis 50

11. DNA Chips 59

12. Career in Biotechnology 64

13. Safeguarding Biodiversity Through Biotechnology 67

4 / Class 12 E d u h e a l F o u n d a tio n

CLASS ­ XI & XII Key Topics in Mathematics for Class XI and XII

I. ALGEBRA

1. Sets, Relations and Functions : Sets and their Representations, Union, intersection and complements of sets, and their algebraic properties, Relations, equivalence relations, mappings, one­one, into and onto mappings, composition of mappings

2 . Complex Numbers : Complex number in the form a + ib and their representation in a plane. Argand diagram. Algebra of complex numbers, Modulous and Arguments (or amplitude) of a complex number, square root of a complex number. Cube roots of unity, triangle – inequality.

3 . Matrices and Determinants : Determinants and matrices of order two and three, properties of determinants, Evaluation of determinants. Area of triangles using determinants, Addition and multiplication of matrices, adjoint and inverse of matrix. Test of consistency and solution of simultaneous linear equations using determinants and matrices.

4 . Quadratic Equations : Quadratic equation in real and complex number system and their solutions. Relation between roots and co­efficients, nature of roots, formation of quadratic equations with given roots; Symmetric functions of roots.

5 . Permutation and Combination : Fundamental principle of counting; Permutation as an arrangement. Meaning of P(n, r) and C (n , r). Simple applications.

6 . Mathematical Induction and Its applications :

7 . Binomial Theorem and its Applications : Binomial Theorem for a positive integral index; general term and middle term; Binomial Theorem for any index. Properties of Binomial Co­efficients. Simple applications for approximations.

8 . Sequences and Series : Arithmetic, Geometric and Harmonic progressions. Special cases of Sn , Sn2, Sn3 . Arithmetic­ Geometric Series, Exponential and Logarithmic series.

II. CALCULUS

9. Differential Calculus : Polynomials, rational, trigonometric, logarithmic and exponential functions, Inverse functions. Graphs of simple functions. Limits, Continuity; differentiation of the sum, difference, product and quotient of two functions. differentiation of trigonometric, inverse trigonometric, logarithmic, exponential, composite and implicit functions; derivatives of order upto three. Applications of derivative: monotonic functions, Maxima and minima of functions of one variable.

10 . Integral Calculus : Integral as an anti­derivative. Fundamental integrals involving algebraic, trigonometric, exponential and logarithmic functions. Integration by substitution, by parts and by partial fractions. Integration using trigonometric identities. Integral as limit of a sum. Properties of definite integrals. Evaluation of indefinite integrals; Determining areas of the regions bounded by simple curves.

11. Differential Equations : Ordinary differential equations, their order and degree. Solution of differential equations by the method of separation of variables. Solution of homogeneous and linear differential equations.

III. TWO AND THREE DIMENSIONAL GEOMETRY

12. Two dimensional Geometry : Recall of Cartesian system of Rectangular co­ordinates in a plane, distance formula, area of a triangle, condition for the collinearity of three points and section formula, centroid and in­centre of a triangle, locus and its equation, translation of axes, slope of a line, parallel and perpendicular lines, intercepts of a line on the coordinate axes. The straight line and pair of straight lines Various forms of equations of a line, intersection of lines, angles between two lines, conditions for concurrency of three lines, distance of point from a line, coordinates of orthocentre and circumcentre of triangle, equation of family of lines passing through the point of intersection of two lines, homogeneous equation of second degree in x and y,

Statements given in italics are related to biotechnology. The syllabus guideline given is only indicative & not exhaustive.

Class 12 / 5 E d u h e a l F o u n d a tio n

angle between pair of lines through the origin, combined equation of the bisectors of the angles between a pair of lines, condition for the general second degree equation to represent a pair of lines, point of intersection and angle between two lines represented by S = O and the factors of S .

Circles and system of Circles Standard form of equation of a circle, general form of the equation of a circle, its radius and centre, equation of a circle in the parametric form, equation of a circle when the end points of a diameter are given, points of intersection of a line and a circle with the centre at the origin and condition for a line to be tangent to the circle, length of the tangent, equation of the tangent, equation of a family of circles through the intersection of two circles, condition for two intersecting circles to be orthogonal.

Conic Section Sections of cones, equations of conic sections (parabola, ellipse and hyperbola) in standard forms, condition for y = mx + c to be a tangent and point(s) of tangency.

13 . Three dimensional Geometry : Coordinates of a point in space, distance between the points; Section formula, direction ratios and direction cosines, angle between two intersecting lines, equations of a line and a plane in different forms; intersection of a line and a plane, coplanar lines, equation of a sphere, its centre and radius. Diameter form of the equation of a sphere.

IV. VECTORS

14. Vector Algebra : Vectors and Scalars, addition of vectors, components of a vector in two dimensions and three dimensional space, scalar and vector products, vector triple product. Application of vectors to plane geometry.

V. STATISTICS

15. Measures of Central Tendency and Dispersion : Calculation of Mean, median and mode of grouped and ungrouped data. Calculation of standard deviation, variance and mean deviation for grouped and ungrouped data.

16 . Probability : Probability of an event, addition and multiplication theorems of probability and their applications; Conditional probability; Probability distribution of a random variable; Binomial distribution and its properties.

VI. TRIGONOMETRY

17. Trigonometrical ratios, identities and equations. Inverse trigonometric functions and their properties. Properties of triangles, solution of triangles. Heights and Distances.

VII. STATICS AND DYNAMICS

18. Statics : Resultant of Coplanar forces; moments and couples. Equilibrium of three concurrent forces.

19 . Dynamics : Speed and velocity, average speed, instantaneous speed, acceleration and retardation, resultant of two velocities, relative velocity and its simple applications. Motion of a particle along a line, moving with constant

acceleration. Motion under gravity. Laws of motion, Projectile motion.

Key Topics in Physics for Class XI and XII 1 . Units and Measurement : Units for measurement, system of units – S.I., fundamental and derived units. Dimensions

and their applications.

2 . Description of Motion in one dimension : Motion in a straight line, uniform motion, its graphical representation. Uniformly accelerated motion, and its applications.

3 . Description of Motion in Two and Three dimensions : Scalars and vectors, vector addition, multiplication of a vector by a real number, zero­vector and its properties. Resolution of vectors. Scalar and vector products, uniform circular motion and its applications, projectile motion.

4 . Laws of Motion : Force and inertia – Newton’s Laws of motion. Conservation of linear momentum, rocket propulsion. Inertial frames of references. Static and kinetic friction, laws of friction, rolling friction.<O:P

5 . Work, Energy and Power : Concept of work, energy and power, Energy – kinetic and potential. Conservation of energy. Elastic collision in one and two dimensions. Different forms of energy.

6 . Rotational Motion and Moment of Inertia : Centre of mass of a two­particle system. Centre of mass of a rigid body, general motion of a rigid body, nature of rotational motion, torque, angular momentum, conservation of angular

6 / Class 12 E d u h e a l F o u n d a tio n

momentum and its applications. Moment of Inertia and its physical significance, parallel and perpendicular axes theorem, expression of moment of inertia for ring, disc and sphere.

7 . Gravitation : Acceleration due to gravity, one and two­dimensional motion under gravity. Universal law of gravitation, variation in the acceleration due to gravity of the earth. Planetary motion, artificial satellite – geostationary satellite, gravitational potential energy near the surface of earth, gravitational potential and escape velocity.

8 . Properties of Matter : Inter­atomic and inter­molecular forces, states of matter (A) Solids : Elastic properties, Hook’s law, Young’s modulus, bulk modulus, modulus of rigidity. (B) Liquids : Cohesion and adhesion. Surface energy and surface tension. Flow of fluids, Bernoulli’s theorem and its applications. Viscosity, Stoke’s Law, terminal velocity. (C) Ideal gas laws.

9 . Oscillations : Periodic motion, simple harmonic motion and its equation of motion, energy in S.H.M., Oscillations of a spring and simple pendulum.

10 . Waves : Wave motion, speed of a wave, longitudinal and transverse waves, superposition of waves, progressive and standing waves, vibration of strings and air­columns, beats, resonance. Doppler effect in sound.

11. Heat and Thermodynamics : Thermal expansion of solids, liquids and gases and their specific heats, relationship between C p and C v for gases, first law of thermodynamics, thermodynamic processes. Second law of thermodynamics, Carnot cycle, efficiency of heat engines.

12 . Transference of heat : Modes of transference of heat. Thermal conductivity. Black body radiations, Kirchoff’s law, Wien’s law, Stefan’s law of radiation and Newton’s law of cooling.

13 . Electrostatics : Charges and their conservation, Coulomb’s law, S.I. unit of charge, dielectric constant, electric field, lines of force, field due to dipole and its behavior in a uniform electric field, electric flux, Gauss’s law in simple geometries. Electric potential, potential due to a point charge. Conductors and insulators, distribution of charge on conductors. Capacitance, parallel plate capacitor, combination of capacitors, energy of capacitor, van de graf generator.

14 . Current Electricity : Current as a rate of flow of charges, sources of energy, cells­primary and secondary, grouping of cells resistance of different materials, temperature dependence, specific resistance, Ohm’s law, Kirchoff’s law, series and parallel circuits. Wheatstone Bridge, measurement of voltages and currents, potentiometer.

15 . Thermal and Chemical Effects of currents : Heating effects of current, electric power, simple concept of thermo electricity – (Seeback effect and its explanation), thermocouple, Chemical effects of current and laws of electrolysis.

16 . Magnetic Effects of Currents : Oersted’s experiment, Biot­Savert’s law (magnetic field due to a current element), magnetic field due to a straight wire, circular loop and solenoid, force on a moving charge in a uniform magnetic field (Lorentz force), forces and torques on currents in a magnetic field, force between two current carrying wires, moving coil galvanometer, ammeter and voltmeter.

17 . Magnetostatics : Bar magnet, magnetic field, lines of force, torque on a bar magnet in a magnetic field, earth’s magnetic field, tangent galvanometer, vibration magnetometer, para, dia and ferro­magnetism, magnetic induction, magnetic susceptibility.

18 . Electromagnetic Induction and Alternating Currents : Induced e.m.f., Farady’s Law, Lenz’s Law, self and mutual induction, alternating currents, impedance and reactance, power in a.c. circuits, LCR series combination, resonant circuits. Transformer, simple motor, and A.C. generator.

19 . Ray Optics : Sources of light, luminous intensity, luminous flux, illuminance and photometry(elementary idea). Reflection and refraction of light at plane and curved surfaces, total internal reflection, optical fibre; deviation and dispersion of light by a prism; Lens formula, magnification and resolving power; microscope and telescope.

20 . Wave Optics : Wave nature of light; Interference – Young’s double slit experiment. Diffraction – diffraction due to a single slit. Elementary idea of polarization, Doppler effect of light.

21 . Electromagnetic waves : Electromagnetic oscillations, Electromagnetic wave spectrum from gamma to radio waves – their uses and propogation properties of the atmosphere w.r.t. electromagnetic spectrum.

22 . Electrons and Photons : Discovery of electrons, cathode rays, charge on an electron, e/m for an electron, photoelectric effect and Einstein’s equation of photoelectric effect.

23 . Atoms, Molecules and Nuclei : Rutherford model of the atom, Bohr’s model, energy quantizations, hydrogen spectrum; Atomic masses, size of the nucleus; Radioactivity; rays and their properties – alpha, beta and gamma decay;

Class 12 / 7 E d u h e a l F o u n d a tio n

half life and mean life of radio­active nuclei, Binding energy, mass energy relationship, nuclear fission and nuclear fusion. 24 . Solids and Semi­Conductor Devices : Energy bands in solids, conductors, insulators and semi­conductors, PN

junction, diodes, diode as rectifier, junction transistor, transistor as an amplifier.

Key Topics in Chemistry for Class XI and XII

1 . Atoms, Molecules and Chemical Arithmetic : Measurement in chemistry (significant figures, SI unit, Dimensional analysis). Chemical classification of matter (mixtures, compounds and elements, and purification). Law of chemical combination and Dalton’s Atomic theory. Atomic Mass (mole concept, determination of chemical formulae). Chemical equation (balancing of chemical equation and calculations using chemical equations).

2 . Elements, their Occurrence and extraction : Earth as a source of elements, elements in biology, extraction of metals (mettallurgical process, production of concentrated ore, production of metals and their purification). Mineral wealth of India. Qualitative test of metals.

3 . States of Matter Gaseous state : (measurable properties of gases, Boyle’s Law, Charle’s Law and absolute scale of temperature, Avogadro’s hypothesis, ideal gas equation, Dalton’s law of partial pressure). Kinetic molecular theory of gases (the microscopic model of gas, deviation form ideal behaviour). The solid state (classification of solids, X­ ray studies of crystal lattices and unit cells, packing of constituent particles in crystals). Liquid state (Properties of liquids, Vapour pressure, Surface tension, Viscosity).

4 . Atomic Structures Constituents of the atom : (Discovery of electron, Rutherford model of the atom). Electronic structure of atoms (nature of light and electromagnetic waves, atomic spectra, Bohr’s model of Hydrogen atom, Quantum mechanical model of the atom, electronic configurations of atoms, Aufbau principle). Dual nature of matter and radiation. The uncertainty principle. Orbitals and Quantum numbers. Shapes of orbitals. Electronic configuration of atoms.

5 . Chemical Families – Periodic Properties : Mendeleev’s Periodic Table, Modern Periodic Law, Types of elements (Representative elements­s & p block elements, inner transition elements – d­block elements, inner transition elements – f­block elements). Periodic trends in properties. (Ionization energy, electron affinity, atomic radii, valence, periodicity in properties of compounds).

6 . Chemical Bonding and Molecular structure : Chemical bonds and Lewis structure shapes of molecules (VSEPR theory). Quantum theory of the covalent bond (Hydrogen and some other simple molecules, carbon compounds, hybridization, Boron and Beryllium compounds). Coordinate covalent bond (Ionic bond as an extreme case of polar covalent bond, ionic character of molecules and polar molecules. Bonding in solid state (Ionic, molecular and covalent solids, metals). Hydrogen bond, Resonance. Molecules: Molecular orbital method. Formation of H 2 , O 2 , N 2 , F 2 on the basis of MOT. Hybridisation, Dipole moment and structure of molecules.

7 . The Solid State : Structure of simple ionic compounds. Close­packed structures. Ionic­radii, Silicates (elementary ideas). Imperfection in solids (point defects only). Properties of solids, Amorphous solids. The Gaseous state : Ideal gas equation­Kinetic theory (fundamentals only)

8 . Solutions : Types of solutions, Vapour­pressure of solutions and Raoult’s law. Colligative properties. Non­ideal solutions and abnormal molecular masses. Mole concept­stoichemistry, volumetric analysis­concentration unit.<O:P

9 . Chemical Energetics and Thermodynamics : Energy changes during a chemical reaction, Internal energy and Enthalpy (Internal energy, Enthalpy, Enthalpy changes, Origin of Enthalpy change in a reaction, Hess’s Law of constant heat summation, numericals based on these concepts). Heats of reactions (heat of neutralization, heat of combustion, heat of fusion and vaporization). Sources of energy (conservation of energy sources and identification of alternative sources, pollution associated with consumption of fuels. The sun as the primary source). First law of thermodynamics: Internal energy, Enthalpy, application of first law of thermodynamics. Second law of thermodynamics : Entropy, Free energy, Spontaneity of a chemical reaction, free energy change and chemical equilibrium, free energy available for useful work.

10 . Chemical Equilibrium : Equilibria involving physical changes (solid­liquid, liquid­gas equilibrium involving dissolution of solid in liquids, gases in liquids, general characteristics of equilibrium involving physical processes). Equilibria involving chemical systems (the law of chemical equilibrium, the magnitude of the equilibrium constant, numerical problems). Effect of changing conditions of systems at equilibrium (change of concentration, change of temperature, effect of catalyst­Le Chateliar’s principle). Equilibria involving ions (ionization of electrolytes, weak and strong electrolytes, acid­base equilibrium, various concepts of acids and bases, ionization of water, pH, solubility product,

8 / Class 12 E d u h e a l F o u n d a tio n

numericals based on these concepts).

11. Redox Reactions and Electrochemistry : Oxidation and reduction as an electron transfer process. Redox reactions in aqueous solutions­electrochemical cells. EMF of a galvanic cell. Dependence of EMF on concentration and temperature (nearest equation and numerical problems based on it). Electrolysis, Oxidation numbers (rules for assigning oxidation number, redox reactions in terms of oxidation number and nomenclature). Balancing of oxidation­reduction equations. Electrolytic conduction. Voltaic cell, Electrode potential and Electromotive force, Gibb’s free energy and cell potential. Electrode potential and Electrolysis.

12 . Rates of Chemical Reactions and Chemical Kinetics : Rate of reaction, Instantaneous rate of reaction and order of reaction. Factors affecting rates of reactions (factors affecting rate of collisions encountered between the reactant molecules, effect of temperature on the reaction rate, concept of activation energy, catalysis). Effect of light on rates of reactions. Elementary reactions as steps to more complex reactions. How fast are chemical reactions?Rate expression. Order of a reaction (with suitable examples). Units of rates and specific rate constants. Order of reaction and effect of concentration. (study will be confined to first order only). Temperature dependence of rate constant – Fast reactions (only elementary idea). Mechanism of reaction (only elementary idea). Photochemical reactions.

13 . Chemistry of Hydrocarbons : Alkanes (structure, isomerism, conformation). Stereo Isomerism and chirality (origin of chirality, optical rotation, racemic mixture) Alkenes (isomerism including cis­trans). Alkynes. Arenes (structure of benzene, resonance structure, isomerism in arenes). Sources of hydrocarbons (origin and composition of coal and petroleum; Hydrocarbons from coal and petroleum cracking and reforming, quality of gasoline­octane number, gasoline additives). Laboratory preparation of alkanes (preparation from unsaturated hydrocarbons, alkyl halides and carboxylic acids). Laboratory preparation of alkenes (preparation from alcohols, alkyl halides). Laboratory prepration of alkynes (preparation from calcium carbide and accetylene). Physical properties of alkanes( boiling and melting points, solubility and density). Reactions of hydrocarbons (oxidation, addition, substitution and miscellaneous reactions).

14 . Purification and Characterisation of Organic Compounds : Purification (crystallization, sublimation, distillation, differential extraction, chromatography). Qualitative analysis (analysis of nitrogen, sulphur, phosphorus and halogens). Quantitative analysis (estimation of carbon, hydrogen, nitrogen, halogens, sulphur, phosphorus and oxygen). Determination of molecular mass (Victor Mayer’s method, volumetric method). Calculation of empirical formula and molecular formula. Numerical problems in organic quantitative analysis, modern methods of structure elucidation.

15 . Organic Chemistry Based on Functional Group : (Halides and Hydroxy compounds) Nomenclature of compounds containing halogen atoms and hydroxyl groups : haloalkanes, haloarenes; alcohols and phenols. Correlation of physical properties and uses. Preparation, properties and uses of following. Polyhalogen compounds : Chloroform, Idoform Polyhydric compounds, Ethane 1, 2­diol; Propane­1,2,3 triol. Structure and reactivity – (a) Induction effect, (b) Mesomeric effect, (c) Electrophiles and Nucleophiles, (d) Types of organic reaction.

16 . Organic Chemistry Based on Functional Group II : (Ethers, aldehydes, ketones, carboxylic acids and their derivatives). Nomenclature of ethers, aldehydes, ketones, carboxylic acids and their derivatives. Sacylhalides, acid anhydrides, amides and esters). General methods of preparation, correlation of physical properties with their structures, chemical properties and uses. (Note : Specific compounds should not be stressed for the purpose of evaluation)

17 . Organic Chemistry Based on Functional Group­II : (Cyanides, isocyanides, nitrocompounds and amines) Nomenclature and classification of amines, cynadies, isocyanides, nitro compounds and their method of preparation; correlation of physical properties with structure, chemical reactions and uses.

18 . Chemistry of Non­metals – (Hydrogen, Oxygen and Nitrogen) Hydrogen (position in periodic table, occurrence, isotopes, properties, reactions and uses) Oxygen (occurrence, preparation, properties and reactions, uses, simple oxides; ozone) Water and hydrogen peroxide (structure of water molecule and its aggregates, physical and chemical properties of water, hard and soft water, water softening, hydrogen peroxides, preparation, properties, structure and uses). Nitrogen(Preparation, properties, uses, compounds of Nitrogen – Ammonia, Oxides of Nitrogen, Nitric Acid – preparation, properties and uses).

19 . Chemistry of Non­metals – II : (Boron, Carbon, Silicon, phosphorus, sulphur, halogens and the noble gases). Boron (occurrence, isolation, physical and chemical properties, borax and boric acid, uses of boron and its compounds). Carbon, inorganic compounds of carbon (oxides, halides, carbides), elemental carbon. Silicon (occurrence, preparation and properties, oxides and oxyacids of phosphorus, chemical fertililzers). Sulphur (occurrence and extraction, properties

Class 12 / 9 E d u h e a l F o u n d a tio n

and reactions, oxides: Sulphuric acid – preparation, properties and uses, sodium thiosulphate). Halogens (occurrence, preparation, properties, hydrogen halides, uses of halogens). Noble gases (discovery, occurrence and isolation, physical properties, chemistry of noble gases and their uses).

20 . Chemistry of lighter Metals : Sodium and Potassium (occurrence and extraction, properties and uses, important compounds – NaCl, Na2CO 3 , NaHCO 3 , NaOH, KCI, KOH). Magnesium and calcium (occurrence and extraction, properties and uses, important compounds MgCl 2 , MgSO 4 , CaO, Ca(OH) 2 , CaCO 3 , CaSO 4 , plaster of paris). Aluminium (occurrence, extraction, properties and uses, compounds – AlCl 3 , alums). Cement. Biological role of Sodium, Potassium, Magnesium and Calcium.

21 . Heavy Metals : Iron (Occurrence and extraction, compounds of iron, oxides, halides, sulphides, sulphate, alloy and steel). Copper, silver and gold (occurrence and extractions, properties and uses, compound – sulphides, halides and sulphates, photography). Zinc and Mercury (occurrence and extraction, properties and uses, compound­oxides, halides; sulphides and sulphates). Tin and Lead (occurrence and extraction, properties and uses, compounds – oxides, sulphides, halides).

22 . Chemistry of Representative Elements : Periodic properties – Trends in groups and periods (a) Oxides­nature (b) Halides­melting points (c) Carbonates and sulphates – solubility. The chemistry of s and p block elements, electronic configuration, general characteristics properties and oxidation states of the following :­ Group 1 elements – Alkali metals Group 2 elements – Alkaline earth metals Group 13 elements – Boron family Group 14 elements – Carbon family Group 15 elements – Nitrogen family Group 16 elements – Oxygen family Group 17 elements – Halogen family Group 18 elements – Noble gases and Hydrogen

23 . Transition Metals including Lanthanides : Electronic configuration: General characteristic properties, oxidation states of transition metals. First row transition metals and general properties of their compounds­oxides, halides and sulphides. General properties of second and third row transition elements (Groupwise discussion). Preparation, properties and uses of Potassium dichromate, Potassium permaganate. Inner Transition Elements: General discussion with special reference to oxidation states and eamthanide contraction.

24 . Coordination Chemistry and Organo Metallics : Coordination compounds, Nomenclature : Isomerism in coordination compounds; Bonding in coordination compounds, Werner’s coordination theory.

25 . Nuclear Chemistry : Nature of radiation from radioactive substances. Nuclear reactions; Radioactive disintegration series; Artificial transmutation of elements; Nuclear fission and Nuclear fusion: Isotopes and their applications: Radio carbon­dating.

26 . Synthetic and Natural Polymers : Classification of Polymers, natural and synthetic polymers (with stress on their general methods of preparation) and important uses of the following : Teflon, PVC, Polystyrene, Nylon­66, terylene Environmental pollution – pollutants – services – check and alternatives.

27 . Surface Chemistry : Surfaces : Adsorption Colloids – (Preparation and general properties), Emulsions, Micelles Catalysis : Homogeneous and heterogeneous, structure of catalyst.

28 . Bio Molecules and Biological Processes : The Cell Carbohydrates : Monosaccharides, Disaccharides, Polysaccharides Amino Acides and Peptides – Structure and classification. Proteins and Enzymes – Structure of Proteins, Role of enzymes. Nucleic Acids – DNA and RNA Biological functions of Nucleic acids – Protein synthesis and replication Lipids – Structure, membranes and their functions.

29 . Chemistry in Action : Dyes, Chemicals and medicines (antipyretic, analgesic, antibiotics & tranquilesers), Rocket propellants.<O:P (Structural formulae non­evaluative)

Key Topics in Biology for Class XI and XII

1 . General Biology : Biology and its branches; relationships with other sciences; scientific methods in Biology; historical breakthroughs; scope of Biology and career options; role of Biology in dispelling myths and misbeliefs; characters of living organisms, (elementary idea of metabolism, transfer of energy at molecular level, open and closed system, homoeostasis, growth and reproduction, adaptation, survival, death).

10 / Class 12 E d u h e a l F o u n d a tio n

2 . Systematics and Classification : Variety of living organisms; Systematics; need, history and types of classifications (ar­ tificial, natural, phylogenetic); biosystematics; binomial nomenclature; Two kingdom sys­tem, Five kingdom system, their merits and demerits, status of bacteria and virus; botanical gardens and herbaria; zoological parks and museums. Salient features of various plant groups; classification of angiosperms up to series level (Bentham and Hooker’s system). Salient features of non­chordates up to phylum level and chordates up to class level).

3 . Animal Kingdom : Classification of animal kingdom, characteristics of different phyla and their example. Morphology of Animals ­ Salient features of earthworm, cockroach and rat; tissue sys­tems, structure and function of tissues ­ epithelial, connective, muscular and nervous.

4 . Plant Kingdom : Classification of plants groups, their characteristics and examples. 5 . Cell Biology : Cell as a basic unit of life — discovery of cell, cell theory, cell as a self­contained unit; prokaryotic and

eukaryotic cell; unicellular and multicellular organisms; tools and techniques (compound microscope, electron microscope and cell fractionation); Ultrastructure of prokaryotic and eukaryotic cell ­ cell wall, cell membrane ­ unit membrane concept (fluid mosaic model); membrane transport; cellular movement (exocytosis, endocytosis); cell organelles and their functions ­ nucleus, mitochondria, plastids, endoplasmic reticulum, Golgi complex, lysosomes, microtubules, centriole, vacuole, cy­ toskeleton, cilia and flagella, ribosomes. Molecules of cell; inorganic and organic materials — water, salt, mineral ions, carbohy­drates, lipids, amino acids, proteins, nucleotides, nucleic acids (DNA and RNA); Enzymes (properties, chemical nature and mechanism of action); vitamins, hormones and steroids. Cell cycle : significance of cell division; amitosis, mitosis and meiosis; karyotype analysis.

6 . Genetics : Continuity of life ­ heredity, variation; Mendel’s laws of inheritance; chromosomal basis of inheritance; other patterns of inheritance ­ incomplete dominance, multiple allelism, quantitative inheritance. Chromosomes ­ bacterial cell and eukaryotic cell; parallelism between genes and chro­mosomes; genome, linkage and crossing over; gene mapping; recombination; sex chro­mosomes; sex determination; sex linked inheritance; mutation and chromosomal aberra­tions; Human genetics ­ methods of study, genetic disorders. DNA as a genetic material ­ its structure and replication; structure of RNA and its role in protein synthesis; Gene expression ­ transcription and translation in prokaryotes and eukaryotes; regulation of gene expression, induction and repression ­ housekeeping genes; nuclear basis of differentiation and develop­ ment; oncogenes. Basics of Recombinant DNA technology; cloning; gene bank; DNA fingerprinting; genomics ­ principles and applications, transgenic plants, animals and microbes.

7 . Human Biology : Nutrition and its types; nutrients ­ food and vitamins; Intracellular and extracellular digestion; digestive system of invertebrate (cockroach); digestive system and process in humans (digestion, ingestion, absorption, assimilation, egestion); role of enzymes and hormones in digestion; malnutrition and undernutrition; disorders related to nutrition. Gaseous exchange in animals (earthworm, cockroach); respiration in humans ­ respiratory organs, mechanism; breathing and its regulation : transport of gases through blood; common respiratory disorders ­ prevention and cure. Circulation of body fluids ­ open system in cockroach; closed system in humans, blood and its composition, structure and pumping action of human heart; pulmonary and systemic circulation; heart beat and pulse; rhythmicity of heart­beat, blood related disorders ­ hypertension, atheroma and arteriosclerosis; ECG; pacemaker; lymphatic system, im­munity and immune system. Nitrog­ enous waste elimination ­ ammonetelism, ureotelism, uricotelism; excretory system of cockroach and humans; composition and formation of urine; role of kidney in osmoregulation, kidney failure; dialysis, kidney transplantation; role of ADH; role of liver in excretion. Locomotion and movements; human skeleton ­ axial and appendicular including cranium and rib cage bones; Joints and their types; bone, cartilage and their disorders (arthritis, osteoporosis); mechanism of muscle contraction; red and white muscles in movements. Nervous coordination in cockroach and humans; human nervous system ­ structure and functions of brain and spinal cord, transmission of nerve impulse; reflex action; sensory receptors; structure and function of sense organs ­ eye, ear, nose and tongue. Human endocrine system; hormones and their functions; hormonal imbalance and diseases; role of hormones as messengers and regulators; hypothalamo ­ hypophysial axis; feedback controls. Types of reproduction ­ a general account (asexual and sexual); human male and female reproductive systems; Reproductive cycle in human female, gametogenesis; Fertilization ­ physical and chemical events; development of zygote upto 3 germinal layers and their derivatives; extra­embryonic membranes; general aspects of placenta. Cellular growth ­ growth rate and growth curve; hormonal control of growth; mechanism and types of regeneration; ageing ­ cellular and extracellular changes; theories of ageing.

8 . Angiosperm Botany : Morhpology ­ root, stem and leaf, their structure and modification; Inflorescence, flower, fruit, seed and their types; Description of Poaceae, Liliaceae, Fabaceae, Solanaceae, Brassicaceae and Asteraceae. Internal structure of plants ­ Tissues (meristematic and permanent); tissue systems; anatomy of root, stem and leaf of monocot and dicot; secondary growth. Cell as a physiological unit; water relations ­ absorption and movement (diffusion, osmosis, plasmolysis, permeability, water potential, imbibition); theories of water translocation ­ root pressure, transpiration pull; transpiration ­ significance, factors af­fecting rate of transpiration; mechanism of stomatal opening and closing (Potassium ion theory).

Class 12 / 11 E d u h e a l F o u n d a tio n

Mineral nutrition ­ functions of minerals, essential major elements and trace elements; deficiency symptoms of elements; translocation of solutes, nitrogen and nitrogen metabolism with emphasis on biological nitrogen fixation. * Photosynthesis ­ significance, site of photosynthesis (functional aspect of chlorophyll structure); photochemical and biosynthetic phases; electron transport system; photophosphorylation (cyclic and non­cyclic); C 3 and C 4 Pathway; photorespiration; factors affecting photosynthesis; mode of nutrition (autotrophic, heterotrophic ­ saprophytic, parasitic and insectivorous plants), chemosynthesis. Mechanism of respiration ­ glycolysis, Krebs cycle, pentose pathway, anaerobic respira­tion; respiratory quotient; compensation point; fermentation. Modes of reproduction in flowering plants ­ vegetative propagation (natural and artificial), significance of vegetative propagation; micropropagation; sexual reproduction ­ develop­ment of male and female gametophytes; pollination (types and factors); double fertilisation, incompatibility, embryo development, parthenogenesis and parthenocarpy. * Characteris­ tics of Plant growth; growth regulators (phytohormones) ­ Auxins, gibberellins, cytokinins, ethylene, ABA; seed germination ­ mechanism and factors affecting germina­tion, role of growth regulators in seed dormancy; senescence; abscission; stress factors (salt and water) and growth; plant movement ­ geotropism, phototropism, turgor growth movements (tropic, nastic and nutation), process of flowering ­ photoperiodism, vernalisation.

9 . Ecology and Environment : Organisms and their environment; factors ­ air, water, soil, biota, temperature and light; range o f tolerance; ecological adaptation. Levels of organisation ­ population, species, community, ecosystem and biosphere; Eco­ logical interactions ­ symbiosis, mutualism, commensalism, parasitism, predation and competition. Ecosystem ­ structure and functions; productivity; energy flow; ecological efficiencies; decomposition and nutrient cycling; major blomes ­ forests, grasslands and deserts. Ecological Succession ­ types and mechanism. Natural resources ­ types, use and misuse of natural resources. Environmental pollution ­ kinds, sources and abatement of air, water, soil and noise pollution. Global environ­ mental changes; Greenhouse gases, global warming, sea level rise and ozone layer depletion. Biotic resources ­ terrestrial and aquatic including marine resources; bio­diversity ­benefits and assessment; threats, endangered species, extinctions; conserva­ tion of bio­diversity (biosphere reserves and other protected areas); National and International efforts ­ both governmental and non­governmental; environmental ethics and legislation.

10 . Application of Biology : Population, environment and development; Population growth and factors ­ (natality, mortality, immigration, emigration, age and sex ratio); impact of population growth; reproductive health; common problems of adolescence (Drugs, Alcohol and Tobacco); social and moral implications; mental and addictive disorders; Risks of indiscrimi­ nate use of drugs and antibiotics; population as a resource. * Food production, breeding, improved varieties, biofertilizers, plant tissue culture and its applications; Brief account of some common crop and animal diseases; biopesticides; genetically modified food; bio­war, biopiracy; biopatent; biotechnology and sustainable agriculture. * Recent advances in vaccines; organ transplantation; immune disorders; modern techniques in disease diagnosis; Elementary knowledge of Haemoglobin estima­ tion and estimation of sugar and urea in blood, TLC, DLC, ESR, lipid profile, ELISA and VIDAL tests; AIDS, STD, cancer (types, causes, diagnosis, treatment); biotechnology in therapeutics ­ hor­mones, interferon and immuno modulations. * Basic concepts of ECG, normal ECG, EEG, CT Scan, MRI and ultrasound.

11 . Evolution : Origin and evolution of Life ­ Oparin­Haldane theory, Miller Experiment; theories of evolution; evidences of evolution; sources of variations (mutation, recombination, genetic drift, migration, natural selection); concept of species; speciation and isolation (geographical and reproductive); origin of species.

*******

Class 12 / 13 E d u h e a l F o u n d a tio n

A wide definition of biotechnology is “any technique that uses living organisms, or substances from those organisms, to make or modify a product, to improve plants or animals, or to develop microorganisms for specific uses”.With this broad definition, one readily sees that biotechnology is not a modern practice, but has been practised for centuries. In the area of agriculture, farmers have been crossing plants to produce hybrids and varieties that are improvements over existing ones. Indeed, the Austrian monk Gregor Mendel, who started the field of genetics over a hundred years ago, did that by crossing pea plants. Enthusiastic gardeners have, over the years, generated over four hundred varieties of the rose plant ­ using the methods or grafting and selecting. Veterinarians and livestock breeders have done similarly with animals. In the field of health, Edward Jenner of Britain realised as early as 1820 that prior infection protected one against the recurrence of smallpox; using this, he vaccinated a dairymaid with previously killed pox virus and provided her with defence against the dreaded disease. The great French scientist Louis Pasteur, who died one hundred years ago, showed how invisible germs spoil milk, wine and cheese, and how the simple act of boiling inactivates them ­ and in this way developed the important technique which goes by his name ­ pasteurisation. He also rescued the wine industry of France from disastrous losses by the introduction of proper strains of yeast, proper conditions of aeration, temperatures and storage and other methods in what today is known as downstream processing. Biotechnology has been intuitively and empirically used in the kitchen for quite some time. Marination, caramelisation, food preservation using naturally occurring substances, pickling, fermentation, tenderising meat using papaya extracts, flavour enhancement using chemicals, gelatinisation, use of skins, bladders and colloidon membranes for selective separation ­ these are some examples of the practice of food biotechnology. Medicine came of age only in the twentieth century. Yet, it owes its present­day sophistication to centuries of a whole host of experiments ­ many of which were crude, tentative and even bizarre, but several of which were curiously successful; later research provided the molecular and technological rationale behind some of these success stories. Some instances are the use of saliva to control bleeding and prevent further infection; liver extracts as health builders for convalescents, the herbal products of India, SouthEast Asia,

BIOTECHNOLOGY AN

INTRODUCTION

14 / Class 12 E d u h e a l F o u n d a tio n

Biot

echn

olog

y Th

roug

h Th

e A

ges

Class 12 / 15 E d u h e a l F o u n d a tio n

2006

Dolly, the first

cloned sheep

died due to

arthiritis.

16 / Class 12 E d u h e a l F o u n d a tio n

China and Korea as antidepressants, anti­hypertensives, and speciality medicines.

The most important advance from the biotechnological point of view came from those in the molecular genetics of microbes about 20 years ago. It became possible to isolate plasmids which are autonomous, non­chromosomal, cyclic DNA molecules found in the cytoplasm of microbial cells. It further became possible to selectively cut and open the circular DNA that constitutes the plasmid through the use of specific enzymes called restriction endonucleases. Soon it also became possible to introduce DNA sequences into linearised (cut) plasmid DNA and reseal (ligate) the circle. This “cut and paste” method has allowed the introduction of foreign DNA into the plasmids. The discovery of restriction enzymes for cuttinig DNA at specific spots and ligases that covalently join DNA molecules through phosphodiester bonds were revolutionary steps in molecular engineering.

Examples Steroids, lipids, sugars, vitamins, coenzymes

Proteins, nucleic acids, polysaccharides

Membranes, cell extracts, chloroplasts, mitochondria

Protoplasts

All types of microbial, plant and animal cells

From plants and animals for use in medicine and surgery

Plants, fruitflies, nematodes, frogs, mice, rats and rabbits

Utilisation Drugs, packaging or encapsulating materials, nutrition, food, cleaning and detergency.

Catalysis, energy sources, copying and reproduction.

Separat ion, energy transduction, in vitro biochemistry

Hybrid cells and cell fusion

Biochemicals, genetic engineering, various purposes of biotechnology.

Biochemicals

Chemicals, biochemicals, toxins, immunochemicals, drug testing transgenics.

System At the level of small molecules

Macromolecular level

Organelle level

Cells with wall removed

Cells

Tissues

Organs and organisms

Practising Biotechnology at various levels

Class 12 / 17 E d u h e a l F o u n d a tio n

Bio­Technology is a research oriented science, a combination of Biology and Technology. It covers a wide variety of subjects like Genetics, Biochemistry, Microbiology, Immunology, Virology, Chemistry and Engineering and is also concerned with many other subjects like Health and Medicine, Agriculture and Animal Husbandry, Cropping system and Crop Management, Ecology, Cell Biology, Soil science and Soil Conservation, Bio­statistics, Plant Physiology, Seed Technology etc.

Biotechnology Tree

BUSINESS APPLICATIONS

Healthcare/ Pharmaceutics Food

Innovations/ Food processing

Plant Agriculture/crop Improvement

Animal agriculture

Fermentation technology

Diagnostics

Energy and Environment/ Management

GENETIC ENGINEERING

PROTEIN ENGINEERING

IMMUNOCHEMISTRY IN VITRO CELL CULTIVATION BIOPROCESS TECHNOLOGY FUNDAMENTAL PRINCIPLES

Human, animal/plant physilogy

Molecular and cell biology

Immunology

Biochemistry

Genetics

Chemical engineering

18 / Class 12 E d u h e a l F o u n d a tio n

Applications of Modern Biotechnology include : • Insect, fungal and virus tolerance – by planting pest resistant crops less chemicals

(pesticides) are used, lowering production costs and reducing the impact on the environment. Examples include potato, maize, cotton and tomato;

• Stress tolerance – increasing the tolerance of crops to extreme stresses such as drought, salt and frost could enable resource poor farmers to produce food in areas where it is most needed.

• Herbicide tolerance – when such crops are planted, more environmentally friendly broad­spectrum herbicides can be used. Examples include rice, cotton and beet;

• Enhanced food value and nutrition – such as changing oil pro­files in oilseed crops, and developing vitamin enriched staple crops such as rice, wheat and corn. Research is also focusing on reducing allergens, and enriching crops with protein.

• Higher yields and greater crop stability – this increases crop production per unit of land; • Control and minimise post harvest losses – this reduces the substantial losses after harvesting, and improves the shelf life of fruits and vegetables, such as tomato, contributing to a higher overall crop yield;

• Reduce the loss of top soil and biodiversity – by promoting low tillage production especially in marginal areas that are not ideal for agriculture;

• Development of improved livestock vaccines – for major diseases affecting productivity, diagnostic tools for disease detection and pedigree verification;

• Impact on small­scale farmers – with potentially large yield impacts and significant financial returns despite higher initial seed costs.

What Are The Benefits of Biotechnology ? Modern biotechnology can make an important contribution to the national priorities of a country in a number of areas: ♦ Enhanced Food security

The promise of biotechnology in food production is its capacity to improve the quality and quantity of plants and animals quickly and effectively. ♦ Improved Health care

In addition to improved health through enhancing the nutritional quality of foods, there are many other uses of modern biotechnology that can further enhance human health: • Inexpensive medicine production – Modern biotechnology is enabling the production of

higher quality drugs at a lower cost; • “Biopharming” – Crops are now being tested as possible delivery systems for

pharmaceuticals, such as banana which could one day contain various vaccines; • Human Genome Project – this research could one day enable genetic diseases to be

understood, diagnosed and perhaps cured; • Gene Therapy – medicines are being developed to target specific cells in the human

body;

Class 12 / 19 E d u h e a l F o u n d a tio n

• HIV/AIDS – The production of vaccines for clinical trials is underway and if successful, the companies undertaking the research could produce the vaccine in large amounts at low cost so they are affordable;

• Forensics and Diagnostics – also known as genetic finger­printing, these techniques could provide invaluable evidence in bringing criminals to justice.

Environmental sustainability In addition to reducing the amount of toxic chemical pesticides that are released into the environment though built in resistance to pests, herbicide resistance means that more environmentally friendly broad­spectrum herbicides can be used to eliminate competing weeds. More novel contributions GM can make towards sustainable development include: • Waste management: “Biomaterials” – biodegradable plastics are being developed using

a micro­organism that degrades polyethylene plastics; • Bioremediation – the use of microorganisms such as bacteria to remove environmental

and often poisonous pollutants from soil and water. Waste cleaning organisms, mainly plants, could be grown at treatment plants and contam­inated areas.

Industrial Development Processes Current GM research is opening up future possibilities which could significantly contribute to national economies, and promote new global collaborations, such as: • Engineering traditional food crops to become valuable industrial crops – e.g. canola is

being used to produce high value industrial oil; • Improved/additional characteristics for processing – such as potatoes that absorb less

oil, and fruits with a longer shelf life, such as tomato; • Transforming raw materials – useful enzymes are now mass produced at low cost and

high quality for various industries; • Biomining – this is the inexpensive extraction of precious metals from low­grade ores

using microbes. Plants are also being developed to mine precious metals, for example Brassica, which concentrate gold from the soil in their leaves.

20 / Class 12 E d u h e a l F o u n d a tio n

Biotechnology is the area of education and research where most of the development has been taking in India and obroad. Some of the acheivements of India (as mentioned by deptt of biotechnology, India) in this field is mentioned as below. Crop Biotechnology : Successful transgenic research has been carried out on banana, cabbage, mustard, mungbean, and wheat for crop improvement, increased nutritional value, disease resistance, and better shelf life, particularly in cotton and rice. Transgenic system of indica rice and wheat have been developed; rice transformed with 3 genes and wheat with one gene to confer stress resistance. A nutritionally important gene AmA1 isolated from Amaranthus has been successfully transferred to potato. India is now a partner in the international rice genome programme with the responsibility to sequence a part of chromosome 11. Bt. cotton trials at several locations have been monitored. The data indicates that Bt. cotton has an average yield advantage of around 4.0 times over non­Bt. cotton. No effects of Bt. protein have been noticed on non­target and beneficial insects. Plant Tissue Culture : Protocol standardization has been completed for a number of species of forest trees, horticulture and plantation crops. Different crosses have been developed in a variety of orchids. Field demonstration trials are being conducted to tissue culture raised coffee, pepper and tea. Complete regeneration systems are now available for 20 different species. Transformation system has been developed for populus species. A polymerase chain reaction (PCR) based molecular diagnostic kit has been perfected for the detection of banana bunchy top virus. The micropropagation Technology parks at TERI, New Delhi and NCL, Pune have produced 10 million plantlets field planted over an area of 6500 ha. at various locations in 17 States. Medicinal and Aromatic Plants : A network of four gene banks has been set up of exsitu conservation of rare, endangered and commercially important medicinal and aromatic plants. About 27,000 stem cuttings of Taxus have been raised and 1500 plants established in natural habitat. Agrotechnology package has been developed for a new variety of Artemisia annua containing 0.5 ­ 1.3% artemisinin and yielding up to 80 kg of artemisinin / ha. An isoquinoline alkaloid, berberin, which acts as an immunomodulatory agent has been identified and isolated from Berbris aristata (Berberry). A herbal product is being developed for burn cases. In vitro bioscreens have been developed for assaying the anti­proliferative and anti­ diabetic medicinal plants. A mission progamme has been initiated for the development of herbal products with the help of biotechnology tools. Bioprospecting and Molecular Taxonomy : Biome maps for North­Eastern and Western Himalayas have been prepared. Taxus and its associated species have been mapped in Talle valley of Arunachal Pradesh. A biomonitoring System has been evolved. The documentation and characterization of ten identified species of the two hot spots has been

BIOTECHNOLOGY DEVELOPMENT IN INDIA

Class 12 / 21 E d u h e a l F o u n d a tio n

completed. Molecular characterization of identified economically important species is being carried out. A cold related gene has been indentified from a plant species in Spiti region of Himachal Pradesh. 23 salt tolerant gene have been isolated and cloned from the mangrove region in Western Ghats. Anti pesticidal fractions have been identified from a tree species for resistance against cotton Boll worm 88% growth inhibition recorded. Agriculturally important bioactive compounds have also been identified from five species of the Kumaon and Garhwal Region. National Bioresource Development Board : Under the national bioresource development Board inventorisation, characterization and sustainable development of the bioresources of various agro­climatic zones and fragile ecosystems of the country has been undertaken. Animal Biotechnology : Transgenic mice expressing neomycin resistance gene, green fluorescent protein gene and other genes have been produced. A buffalo chromosome probe for sex determination of embryo is being applied in the field. Rabies vaccine for animals using vero cell culture is being validated before commercialisation. Conjugates are being evaluated in female dogs for the efficacy of recombinant dog zona pellucida glycoprotein to regulate fertility. Aquaculture and Marine Biotechnology : A PCR based diagnostic method for white spot syndrome virus of shrimp and vaccines for fish pathogens against infection of Aeromonas sp. and pseudomonas sp. have been developed. A process has been perfected for the preparation of pathogen free fermented silkworm pupae silage to act as an ingredient in the animal feed. A feed unit with a capacity of 250 kg pellets per hour has been established at central institute of freshwater Aquaculture. Some bioactive compounds from marine organisms are under development. Medical Biotechnology : A therapeutic immunomodulator for leprosy is now available as Leprovac; in the market. Genetically modified live cholera vaccine has completed Phase ­ I clinical trials in humans with no side effects. The anti­fertility vaccine to control fertility in women has been found to produce specific antibodies at a desired level. Steps are being taken for pilot scale production of rotaviral candidate vaccines for Phase ­ I clinical trials in the country. For Japanese Encephalitis, efforts are on to produce the candidate vaccine to serve as a replacement for the mouse brain derived JE vaccine. A DNA sequence has been identified for its potential use as a vaccine for Rabies. A recombinant BCG carrying a gene for Microbium tuberculosis has been developed. The recombinant proteins and peptide based candidate vaccines are awaiting clinical testing for malaria. Fourteen diagnostic technologies like IgM, Mac ELISA assay systems for detection of dengue, Japanese Encephalitis & West Nile; ELISA system to measure alpha­fetoprotein levels in women; IgM based test system for detection of hepatitis­’A’ virus and urine based ELISA systems for detection of four reproductive hormones have been validated. Two diagnostic test systems developed for HIV have been transferred to industry. Goat hepatocytes were found at deccan medical college, Hyderabad to be more compatible as

22 / Class 12 E d u h e a l F o u n d a tio n

compared to porcine hepatocytes for transplantation in patients with acute fuiminate hepatic failures in humans. Human genetics and genome Analysis : Novel mutations in gene responsible for retino blastoma at L.V. Prasad Eye Institute, Hyderbad provide help in screening and counseling of patients and their affected families. A multiplex polymerase chain reaction (PCR) is being used to provide diagnosis and counseling in Spinocerebellar ataxias, Sixteen genetic diagnosis cum counselling units have been set up in the country for prenatal diagnosis and counseling for major genetic disorders. Almost fourteen thousand affected families have benefited from this programme. Around 2000 tribals have been screened for detecting various genetic disorders. A National Bioethics committee has formulated ethical policies for research on human genetics and genomics. Microbial and Industrial : Several microbial strains have been isolated for leaching of metal ores of Copper, Zinc and Gold, Microbial polysaccharide gellan and xanthan have been produced in high concentration using Sphingomonas sp. Production of cyclodextrin has been standardised using microbial strains of Bacillus firmes and Klebsiella pneumoniae. Technology for the bioremediation of Manganese and Zinc mine dumps has been developed through integrated biotechnological approach. Technologies for the production of Xylanase, inulinase, lipase and protease have been developed. Patents on cephalosporin process and rifimycin­shuttle vectors have been obtained in India and USA. For xanthan gum, an indigenous production process as an import substitute developed at Birla Institute of Scientific Research, Jaipur has been transferred to Sriram Biotech Ltd., Hyderabad. A programme on microbial biodiversity for accession of important microorganisms has been initiated. Biological Pest Control : Technologies for the mass production of candidate biocontrol agents, baculovirus, parasites, predators, antagonists, fungi and bacteria for economically important crops have been transferred to industry. The important crops include coffee, cotton, oilseeds, pulse, spices, sugarcane, tea and vegetables. A crop area of 65,000 ha has been covered under biocontrol/biopesticide efficacy demonstration. Use of twelve candidate biopesticides has been successfully demonstrated in different agro­climatic zones. Baculovirus based technology has been developed for managment of forest insect pest (teak defoliator). Integrated Pest Management module has been developed for green (organic) cotton under irrigated conditions. Fermentation based technologies of three fungal biocontrol agents have been transferred to industry. New potential biopesticide technologies are being revalidated for commercialization. Biofertilisers : Mass production technologies for Azotobacter, Azospirillum, blue green algae (BGA), Mycorrhiza and Rhizobium have been developed and transferred to industry for production and marketing. It has been found that rhizobium biofertiliser can save upto 25­30 kg chemical nitrogen in pulses and leguminous oil seeds. Azotobacter, Azospirillum and Mycorrhiza can make phosphate available in soluble form with other micronutrients to most of the commercial crops. Around 10,000 demonstration programmes benefiting about 35000 farmers have been conducted.

Class 12 / 23 E d u h e a l F o u n d a tio n

Biodiversity Conservation and Environment : Technology package for eco­restoration of mine spoil dumps and phytoremediation of dye industry effluent has been standardized. An efficient crude oil and oil sludge degrading bacterial consortium ‘oil zapper’ and biobeneficiation and desulphurisation technologies have been transferred to the industry. Seribiotechnology : A programme on silkworm genome has been initiated at the centre for DNA finger printing and diagnostics, Hyderabad. A technique has been standardised for effective degumming of silk using fungal protease enzyme for better lusture and softness of dupion silk. Field trials of immunodiagnostic kits have been conducted at farmers’ level for early detection of infectious flatcherie disease. More than 100 microsatellite DNA markers have been identified to be used for mapping genes of silkworm. Eight molecular RFLP markers linked to the cocoon shell character of the silkworm have been identified. They are likely to be used in directional breeding programme. Expression of marker genes has been demonstrated in silkworm. Silkworm is likely to be used as a bioreactor in future. Gynogenic haploids have been produced in mulberry and are being field evaluated. Food biotechnology : Low cost nutritious food supplements for school going children have been developed. Large scale production of betalains (food colorant) and Oyster mushroom optimized. Process for preparation of clarified fruit juices with aroma retention and food flavours developed. Diagnostic kits for rapid detection of food borne pathogens have been developed. Repositories and Biotech Facilities : A large number of infrastructure facilities and repositories have been set up in different universities and research institutes to facilitate advance research. The facility at IMTECH, Chandigarh for the microbial type culture collection has now been upgraded to an International Depository Authority (IDA). The repository on Drosophila has been strengthened to develop educational kits for demonstrating genetic experiments in schools and colleges. Studies at the repository on filaria have resulted in patenting of a unique filarial experiments in schools and colleges. Studies at the repository of filaria have resulted in patenting of a unique filarial antigen. Genetic engineering and strain manipulation has demonstrated over production of daunorubicin and anti­cancer antibiotic at pilot scale. A national containment facility has been set up at NBPGR, New Delhi to look into various quarantine issue relating to transgenic planting materials. A National facility for Virus diagnosis and Quality control in Tissue culture raised planting materials has been set up at Indian Agricultural Research Institute, New Delhi with Six satellite centres. This facility will certify all domestic and export consignments. Bioinformatics : India is one of the first countries in the world to initiate a Bioinformatics programme. A national bioinformatics network has been evolved for sustainable utilization of the biological data resource. The 56 centres, six interactive computer graphics facilities and four post graduate diploma courses in Bioinformatics and being supported. Internationally recognized databases such as EMBnet. PDB, GDB, EBI and plant genome database have been established in the form of mirror sites under the national Jai Vigyan S&T mission. These databases are being utilized for genomic and proteomic R&D activities through high speed and high bandwidth network services.

24 / Class 12 E d u h e a l F o u n d a tio n

Our bodies are filled with intricate, active molecular structures. When these structures are damaged, health suffers. Modern medicine can affect the workings of the body in many ways, but from a molecular viewpoint it remains crude indeed. Molecular manufacturing can construct a range of medical instruments and devices with far greater abilities. The body is an enormously complex world of molecules. With nanotechnology to help, we can learn to repair it. The Molecular Body To understand what nanotechnology can do for medicine, we need a picture of the body from a molecular perspective. The human body can be seen as a workyard, construction site, and battle ground for molecular machines. It works remarkably well, using systems so complex that medical science still doesn’t understand many of them. Failures, though, are all too common. The Body As Workyard Molecular machines do the daily work of the body. When we chew and swallow, muscles drive our motions. Muscle fibers contain bundles of molecular fibers that shorten by sliding past one another. In the stomach and intestines, the molecular machines we call digestive enzymes break down the complex molecules in foods, forming smaller molecules for use as fuel or as building blocks. Molecular devices in the lining of the digestive tract carry useful molecules to the bloodstream. Meanwhile, in the lungs, molecular storage devices called haemoglobin molecules pick up oxygen. Driven by molecular fibers, the heart pumps blood laden with fuel and oxygen to cells. In the muscles, fuel and oxygen drive contraction based on sliding molecular fibers. In the brain, they drive the molecular pumps that charge nerve cells for action. In the liver, they drive molecular machines that build and break down a whole host of molecules. And so the story continues through all the work of the body. Yet each of these functions sometimes fails, whether through damage or inborn defect. The Body As Construction Site In growing, healing, and renewing tissue, the body is a construction site. Cells take building materials from the bloodstream. Molecular machinery programmed by the cell’s genes uses these materials to build biological structures: to lay down bone and collagen, to build

NANOBIOTECHNOLOGY

Class 12 / 25 E d u h e a l F o u n d a tio n

whole new cells, to renew skin, and to heal wounds. With the exception of tooth fillings and other artificial implants, everything in the human body is constructed by molecular machines. These molecular machines build molecules, including more molecular machines. They clear away structures that are old or out of place, sometimes using machinery like digestive enzymes to take structures apart. During tissue construction, whole cells move about, Amoeba like: extending part of themselves forward, attaching, pulling their material along, and letting go of the former attachment site behind them. Individual cells contain a dynamic pattern of molecules made of components that can break down but can also be replaced. Some molecular machines in the cell specialized in digesting molecules that show signs of damage, allowing them to be replaced by fresh molecules made according to genetic instructions. Components inside cells form their complex patterns by self­assembly, that is, by sticking to the proper partners. Failures in construction increase as we age. Teeth wear and crack and aren’t replaced; hair follicles stop working; skin sags and wrinkles. The eye’s shape becomes more rigid, ruining close vision. Younger bodies can knit together broken bones quickly, making them stronger than before, but osteoporosis can make older bones so fragile that they break under minor stress. Sometimes construction is botched from the beginning due to a missing or defective genetic code. In haemophilia, bleeding fails to stop due to the lack of blood clotting factor. Construction of muscle tissue is disrupted in 1 in 3,300 male births by muscular dystrophy, in which muscles are gradually replaced by scar tissue and fat; the molecule “dystrophin” is missing. Sickle cell anemia results from abnormal haemoglobin molecules. Paraplegics and quadriplegics know that some parts of the body don’t heal well. The spinal cord is an extreme—and extremely serious—case, but scarring and improper re­growth of tissues result from many accidents. If tissues always regrew properly, injury would do no permanent physical damage. The Body As Battlefield Assaults from outside the body turn it into a battlefield where the aggressors sometimes get the upper hand. From parasitic worms to protozoa to fungi to bacteria to viruses, organisms of many kinds have learned to live by entering the body and using their molecular machinery to build more of themselves from the body’s building blocks. To meet this onslaught, the body musters the defenses of the immune system—an armada of its own molecular machines. Your body’s own amoebalike white blood cells petrol the bloodstream and move out into tissues, threading their way between other cells, searching for invaders. How can the immune system distinguish the hundreds kinds of cells that should be in the body from the invading cells and viruses that shouldn’t? This has been the central question of the complex science of immunology. The answer, as yet only partially understood, involves a complex interplay of molecules that recognize other molecules by sticking to them in a selective fashion. These include free­floating antibodies—which are a bit like bumbling guided missiles—and similar molecules that are bound to the surface of white

26 / Class 12 E d u h e a l F o u n d a tio n

blood cells and other cells of the immune system, enabling them to recognize foreign surfaces on contact. This system makes life possible, defending our bodies from the fate of meat left at room temperature. Still, it lets us down in two basic ways. First, the immune system does not respond to all invaders, or responds inadequately. Malaria, tuberculosis, herpes, and AIDS all have their strategies for evading destruction. Cancer is a special case in which the invaders are altered cells of the body itself, sometimes successfully masquerading as healthy cells and escaping detection. Second, the immune system sometimes over responds, attacking cells that should be left alone. Certain kinds of arthritis, as well as lupus and rheumatic fever, are caused by this mistake. Between attacking when it shouldn’t and not attacking when it should, the immune system often fails, causing suffering and death. Medicine Today When the body’s working, building, and battling goes awry, we turn to medicine for diagnosis and treatment. Today’s methods, though, have obvious shortcomings. Crude Methods Diagnostic procedures vary widely, from asking a patient questions, through looking at X­ray shadows, through exploratory surgery and the microscopic and chemical analysis of materials from the body. Doctors can diagnose many ills, but others remain mysteries. Even a diagnosis does not imply understanding: doctors could diagnose infections before they knew about germs, and today can diagnose many syndromes with unknown causes. After years of experimentation and untold loss of life, they can even treat what they don’t understand—a drug may help, though no one knows why. Leaving aside such therapies as heating, massaging, irradiating, and so forth, the two main forms of treatment are surgery and drugs. From a molecular perspective, neither is sophisticated. Surgery is a direct, manual approach to fixing the body, now practiced by highly trained specialists. Surgeons sew together torn tissues and skin to enable healing, cut out cancer, clear out clogged arteries, and even install pacemakers and replacement organs. It’s direct, but it can be dangerous: anesthetics, infections, organ rejection, and missed cancer cells can all cause failure. Surgeons lack fine­scale control. The body works by means of molecular machines, most working inside cells. Surgeons can see neither molecules nor cells, and can repair neither. Drug therapies affect the body at the molecular level. Some therapies—like insulin for diabetics—provide materials the body lacks. Most—like antibiotics for infections—introduce materials no human body produces. A drug consists of small molecules in our simulated molecular world, many would fit in the palm of your hand. These molecules are dumped into the body (sometimes directed to a particular region by a needle or the like), where they mix and wander through blood and tissue. They typically bump into other molecules of all sorts in all places, but only stick to and affect molecules of certain kinds. Antibiotics like penicillin are selective poisons. They stick to molecular machines in bacteria

Class 12 / 27 E d u h e a l F o u n d a tio n

and jam them, thus fighting infection. Viruses are a harder case because they are simpler and have fewer vulnerable molecular machines. Worms, fungi, and protozoa are also difficult, because their molecular machines are more like those found in the human body, and hence harder to jam selectively. Cancer is the most difficult of all. Cancerous growth consist of human cells, and attempts to poison the cancer cells typically poison the rest of the patient as well. Other drug molecules bind to molecules in the human body and modify their behaviour. Some decrease the secretion of stomach acid, others stimulate the kidneys, many affect the molecular dynamics of the brain. Designing drug molecules to bind to specific targets is a growth industry today, and provides one of the many short­term payoffs that is spurring developments in molecular engineering. Limited Abilities Current medicine is limited both by its understanding and by its tools. In many ways, it is still more an art than a science. Mark Pearson of Du Pont points out, “In some areas, medicine has become much more scientific, and in others not much at all. We’re still short of what I would consider a reasonable scientific level. Many people don’t realize that we just don’t know fundamentally how things work. It’s like having an automobile, and hoping that by taking things apart, we’ll understand something of how they operate. We know there’s an engine in the front and we know it’s under the hood, we have an idea that it’s big and heavy, but we don’t really see the rings that allow pistons to slide in the block. We don’t even understand that controlled explosions are responsible for providing the energy that drives the machine.” Better tools could provide both better knowledge and better ways to apply that knowledge for healing. Today’s surgery can rearrange blood vessels, but is far too coarse to rearrange or repair cells. Today’s drug therapies can target some specific molecules, but only some, and only on the basis of type. Doctors today can’t affect molecules in one cell while leaving identical molecules in a neighbouring cell untouched because medicine today cannot apply surgical control to the molecular level. Nanotechnology in Medicine Working Outside Tissues Developments in nanotechnology will result in improved medical sensors. As protein chemist Bill DeGrado notes, “Probably the first use you may see would be in diagnostics: being able to take a tiny amount of blood from somebody, just a pinprick, and diagnose for a hundred different things. Biological systems are already able to do that, and I think we should be able to design molecules or assemblies of molecules that mimic the biological system.”

. ; . , 1988.

28 / Class 12 E d u h e a l F o u n d a tio n

In the longer term, though, the story of nanotechnology in medicine will be the story of extending surgical control to the molecular level. The easiest applications will be aids to the immune system, which selectively attack invaders outside tissues. More difficult applications will require that medical nanomachines mimic white blood cells by entering tissues to interact with their cells. Further applications will involve the complexities of molecular­level surgery on individual cells. As we look at how to solve various problems, you’ll notice that some that look difficult today will become easy, while others that might seem easier turn out to be more difficult. The seeming difficulty of treating disorders is always changing: Once polio was frequent and incurable, today it is easily prevented. Syphilis once caused steady physical decline leading to insanity and death; now it is cured with a shot. Athlete’s foot has never been seen as a great scourge, yet it remains hard to cure. Likewise with the common cold. This pattern will continue: Deadly diseases may be easily dealt with, while minor ills remain incurable, or vice versa. As we will see, a mature nanotechnology­based medicine will be able to deal with almost any physical problem, but the order of difficulty may be surprising. Nature cares nothing for our sense of appropriateness. Working Outside Tissues One approach to nanomedicine would make use of microscopic mobile devices built using molecular­manufacturing equipment. These would be biodegradable, more difficult to develop than simple, fixed­location nanomachines, yet clearly feasible and useful. Development will start with the simpler applications, so let’s begin by looking at what can be done without entering living tissues. The skin is the body’s largest organ, and its exposed position subjects it to a lot of abuse. This exposed position, though, also makes it easier to treat. Among the earlier applications of molecular manufacturing may be those popular, quasimedical products, cosmetics. A cream packed with nanomachines could do a better and more selective job of cleaning than any product can today. It could remove the right amount of dead skin, remove excess oils, add missing oils, apply the right amounts of natural moisturizing compounds, and even achieve the elusive goal of “deep pore cleaning” by actually reaching down into pores and cleaning them out. The cream could be a smart material with smooth­on, peel­off convenience. The mouth, teeth, and gums are amazingly troublesome. Today, daily dental care is an endless cycle of brushing and flossing, of losing ground to tooth decay and gum disease as slowly as possible. A mouthwash full of smart nanomachines could do all that brushing and flossing do and more, and with far less effort—making it more likely to be used. This mouthwash would identify and destroy pathogenic bacteria while allowing the harmless flora of the mouth to flourish in a healthy ecosystem. Further, the devices would identify particles of food, plaque, or tartar, and lift them from teeth to be rinsed away. Being

Class 12 / 29 E d u h e a l F o u n d a tio n

suspended in liquid and able to swim about, devices would be able to reach surfaces beyond reach of toothbrush bristles or the fibers of floss. As short­lifetime medical nanodevices, they could be built to last only a few minutes in the body before falling apart into materials of the sort found in foods (such as fiber). With this sort of daily dental care from an early age, tooth decay and gum disease would likely never arise. If under way, they would be greatly lessened. Going beyond this superficial treatment would involve moving among and modifying cells. Let’s consider what can be done with this treatment inside the body, but outside the body’s tissues. The bloodstream carries everything from nutrients to immune­system cells, with chemical signals and infectious organisms besides. Here, it is useful to think in terms of medical nanomachines that resemble small submarines, like the ones in Figure. Each of these is large enough to carry a nanocomputer as powerful as a mid­1980s mainframe, along with a huge database (a billion bytes), a complete set of instruments for identifying biological surfaces, and tools for clobbering viruses, bacteria, and other invaders. Immune cells, as we’ve seen, travel through the bloodstream checking surfaces for foreignness and—when working properly—attacking and eliminating what should not be there. These immune machines would do both more and less. With their onboard sensors and computers, they will be able to react to the same molecular signals that the immune system does, but with greater discrimination. Before being sent into the body on their search­and­destroy mission, they could be programmed with a set of characteristics that lets them clearly distinguish their targets from everything else. The body’s immune system can respond only to invading organisms that had been encountered by that individual’s body. Immune machines, however, could be programmed to respond to anything that had been encountered by world medicine. Immune machines can be designed for use in the bloodstream or the digestive tract (the mouthwash described above used these abilities in hunting down harmful bacteria). They could float and circulate, as antibiotics do, while searching for intruders to neutralize. To escape being engulfed by white blood cells making their own patrols, immune machines could display standard molecules on their surface­molecules the body knows and trusts already—like a fellow police officer wearing a familiar uniform. When an invader is identified, it can be punctured, letting its contents spill out and ending its effectiveness. If the contents were known to be hazardous by themselves, then the immune machine could hold on to it long enough to dismantle it more completely. How will these devices know when it’s time to depart? If the physician in charge is sure the task will be finished within, say, one day, the devices prescribed could be of a type designed to fall apart after twenty­four hours. If the treatment time needed is variable, the physician could monitor progress and stop action at the appropriate time by sending a specific molecule—aspirin perhaps, or something even safer—as a signal to stop work. The inactivated devices would then be cleared out along with other waste eliminated from the body.

30 / Class 12 E d u h e a l F o u n d a tio n

BIOINFORMATICS In the last few decades, advances in molecular biology and the equipment available for research in this field have allowed the increasingly rapid sequencing of large portions of the genomes of several species. In fact, to date, several bacterial genomes, as well as those of some simple eukaryotes (e.g., Saccharomyces cerevisiae, or baker’s yeast) have been sequenced in full. The Human Genome Project, designed to sequence all 24 of the human chromosomes, is complete. Popular sequence databases, such as GenBank and EMBL, have been growing at exponential rates. This deluge of information has necessitated the careful storage, organization and indexing of sequence information. Information science has been applied

to biology to produce the field called Bioinformatics. The simplest tasks used in bioinformatics concern the creation and maintenance of databases of biological information. Nucleic acid sequences (and the protein sequences derived from them) comprise the majority of such databases. While the storage and organization of millions of nucleotides is far from trivial, designing a database and developing an interface whereby researchers can both access existing information and submit new entries is only the beginning. The most pressing tasks in bioinformatics involve the analysis of sequence information. Computational Biology is the name given to this process, and it involves the following: • Finding the genes in the DNA sequences of various organisms • Developing methods to predict the structure and/or function of newly discovered proteins

and structural RNA sequences. • Clustering protein sequences into families of related sequences and the development

of protein models. • Aligning similar proteins and generating phylogenetic trees to examine evolutionary

relationships. The process of evolution has produced DNA sequences that encode proteins with very specific functions. It is possible to predict the three­dimensional structure of a protein using algorithms that have been derived from our knowledge of physics, chemistry and most importantly, from the analysis of other proteins with similar amino acid sequences. The diagram below summarizes the process by which DNA sequences are used to model

Ligand Binding Domain of Retinoic Acid Receptor

Class 12 / 31 E d u h e a l F o u n d a tio n

protein structure. The processes involved in this transformation are detailed below. • Searching for Genes The collecting, organizing and indexing of sequence information into a database, a challenging task in itself, provides the scientist with a wealth of information, albeit of limited use. The power of a database

comes not from the collection of information, but in its analysis. A sequence of DNA does not necessarily constitute a gene. It may constitute only a fragment of a gene or alternatively, it may contain several genes. Luckily, in agreement with evolutionary principles, scientific research to date has shown that all genes share common elements. For many genetic elements, it has been possible to construct consensus sequences, those sequences best representing the norm for a given class of organisms (e.g, bacteria, eukaroytes). Common genetic elements include promoters, enhancers, polyadenylation signal

sequences and protein binding sites. These elements have also been further characterized into further subelements.

Genetic elements share common sequences, and it is this fact that allows mathematical algorithms to be applied to the analysis of sequence data. A computer program for finding genes will contain at least those elements whch are given in table. • The Creation of Sequence Databases Most biological databases consist of long strings of nucleotides (guanine, adenine, thymine, cytosine and uracil) and/or amino acids (threonine, serine, glycine, etc.). Each sequence of nucleotides or amino acids represents a particular gene or protein (or section thereof), respectively. Sequences are represented in shorthand, using single letter designations. This decreases the space necessary to store information and increases processing speed for analysis.

Elements of a Gene­seeking Computer Program

Algorithms for Probability formulae are used to pattern recognition determine if two sequences are

statistically similar.

Data tables These tables contain information on consensus sequences for various genetic elements. More information enables a better analysis.

Taxonomic Consensus sequences vary between differences different taxonomic classes of organisms.

Inclusion of these differences in an analysis speeds processing and minimizes error.

Analysis rules These programming instructions define how algorithms are applied. They define the degree of similarity accepted and whether entire sequences and/or fragments thereof will be considered in the analysis. A good program design enables users to adjust

32 / Class 12 E d u h e a l F o u n d a tio n

While most biological databases contain nucleotide and protein sequence information, there are also databases which include taxonomic information such as the structural and biochemical characteristics of organisms. The power and ease of using sequence information has however, made it the method of choice in modern analysis. In the last three decades, contributions from the fields of biology and chemistry have facilitated an increase in the speed of sequencing genes and proteins. The advent of cloning technology allowed foreign DNA sequences to be easily introduced into bacteria. In this way, rapid mass production of particular DNA sequences, a necessary prelude to sequence determination, became possible. Oligonucleotide synthesis provided researchers with the ability to construct short fragments of DNA with sequences of their own choosing. These oligonucleotides could then be used in probing vast libraries of DNA to extract genes containing that sequence. Alternatively, these DNA fragments could also be used in polymerase chain reactions to amplify existing DNA sequences or to modify these sequences. With these techniques in place, progress in biological research increased exponentially. For researchers to benefit from all this information, however, two additional things were required: 1) ready access to the collected pool of sequence information and 2) a way to extract from this pool only those sequences of interest to a given researcher. Simply collecting, by hand, all necessary sequence information of interest to a given project from published journal articles quickly became a formidable task. After collection, the organization and analysis of this data still remained. It could take weeks to months for a researcher to search sequences by hand in order to find related genes or proteins. Computer technology has provided the obvious solution to this problem. Not only can computers be used to store and organize sequence information into databases, but they can also be used to analyze sequence data rapidly. The evolution of computing power and storage capacity has, so far, been able to outpace the increase in sequence information being created. Theoretical scientists have derived new and sophisticated algorithms which allow sequences to be readily compared using probability theories. These comparisons become the basis for determining gene function, developing phylogenetic relationships and simulating protein models. The physical linking of a vast array of computers in the 1970’s provided a few biologists with ready access to the expanding pool of sequence information. This web of connections, now known as the Internet, has evolved and expanded so that nearly everyone has access to this information and the tools necessary to analyze it. The Challenge of Protein Modelling There are a series of steps following the location of a gene locus to the realization of a three­dimensional model of the protein that it encodes. Step 1 : Location of Transcription Start/Stop A proper analysis to locate a genetic locus will usually have already pinpointed at least the approximate sites of the transcriptional start and stop. Such an analysis is usually sufficient in determining protein structure. It is the start and end codons for translation that must be determined with accuracy.

Class 12 / 33 E d u h e a l F o u n d a tio n

Step 2 : Location of Translation Start/Stop The first codon in a messenger RNA sequence is almost always AUG. While this reduces the number of candidate codons, the reading frame of the sequence must also be taken into consideration. There are six reading frames possible for a given DNA sequence, three on each strand, that must be considered, unless further information is available. Since genes are usually transcribed away from their promoters, the definitive location of this element can reduce the number of possible frames to three. There is not a strong consensus between different species surrounding translation start codons. Therefore, location of the appropriate start codon will include a frame in which they are not apparent abrupt stop codons. Knowledge of a protein Os predicted molecular mass can assist this analysis. Incorrect reading frames usually predict relatively short peptide sequences. Therefore, it might seem deceptively simple to ascertain the correct frame. In bacteria, such is frequently the case. However, eukaryotes add a new obstacle to this process: INTRONS! Step 3 Detection of Intron/Exon Splice Sites In eukaryotes, the reading frame is discontinuous at the level of the DNA because of the presence of introns. Unless one is working with a cDNA sequence in analysis, these introns must be spliced out and the exons joined to give the sequence that actually codes for the protein. Intron/exon splice sites can be predicted on the basis of their common features. Most introns begin with the nucleotides GT and end with the nucleotides AG. There is a branch sequence near the downstream end of each intron involved in the splicing event. There is a moderate consensus around this branch site. Step 4 : Prediction of 3­D Structure With the completed primary amino acid sequence in hand, the challenge of modelling the three­dimensional structure of the protein awaits. This process uses a wide range of data and CPU­intensive computer analysis. Most often, one is only able to obtain a rough model of the protein, and several conformations of the protein may exist that are equally probable. The best analysis will utilize data from all the following sources. All of this information is used to determine the most probable locations of the atoms of the protein in space and bond angles. Graphical programs can then use this data to depict a three­dimensional model of the protein on the two­dimensional computer screen.

...AG CGCG TGAGT..AG..

Introns

Exons

Pattern Alignment to known Comparison homologues whose confor­

mation is more secure

X­ray Diffraction Most ideal when some data is Data available on the protein of

interest. However, diffraction data from homologous proteins is also very valuable.

Physical Forces Biophysical data and analyses /Energy States of an amino acid sequence can

be used to predict how it will fold in space.

34 / Class 12 E d u h e a l F o u n d a tio n

It’s no secret that eating right is the way to stay healthy. But soon the blurry line between foods and medicines may be erased altogether. Research is underway to use staple foods to deliver inexpensive, effective vaccines for specific illnesses — literally, “edible vaccines,” which could save lakhs of children who die each year from preventable diseases. For example, researchers are experimenting with building a vaccine for hepatitis B, which attacks the liver, into bananas. When eaten, the vaccine is absorbed through the intestine into the bloodstream, producing antibodies in the same way as an injected vaccine. But the banana vaccine is expected to cost about Rs. 2 a dose, rather than Rs. 125 for an injection. Plus, it could be easily administered without the need for refrigeration or medical staff. These plant­based vaccines seem to respond directly to a challenge issued centuries ago by Hippocrates, the Greek physician considered to be the father of modern medicine. He said, “Let food be your medicine and medicine be your food.” This figure illustrate the transgenetic implantation to create an edible vaccine. This is done by creating genetically engineered bananas that carry proteins from disease causing organisms. One disease that such a vaccine might protect against is the often fatal diarrhoea caused by the cholera bacterium. Here is how the process works :

Cutting and pasting A strand of the cholera DNA is spliced into a ring of DNA from a benign E.coli bacterium. With the help of an electric pulse, the ring is inserted into as E.coli cell. The cell multiplies, creating millions of copies of the DNA. The modified DNA is spliced again, this time into the DNA ring of a single­celled organism called Agrobacterium, which naturally infects bananas and other plants. When the Agrobacterium attacks a banana cell, the bacterial DNA is introduced into the plant cell and causes it to create proteins like those found in the original cholera bacterium. Divide and conquer Now the banana cell is cloned it divides into may seedling cells, with each new daughter cell inheriting the modified DNA and grows into a complete banana plant. When bananas from this plant are eaten, the cholera proteins cause the body to respond as if it were being invaded by the cholera bacterium. The immune system reacts, providing immunity to the original disease.

INCREDIBLE EDIBLE VACCINES

Class 12 / 35 E d u h e a l F o u n d a tio n

Why a banana? They need not be refrigerated, and are usually eaten raw. Medicinal bananas might even sport a differently colored peel thanks to engineered pigmentation genes.

36 / Class 12 E d u h e a l F o u n d a tio n

DNA computers can’t be found at your local electronics store yet. The technology is still in development, and didn’t even exist as a concept a decade ago. In 1994, Leonard Adleman introduced the idea of using DNA to solve complex mathematical problems. Adleman is often called the inventor of DNA computers (see cartoon). His article in a 1994 issue of the journal Science outlined how to use DNA to solve a well­known mathematical problem, called the directed Hamilton Path problem, also known as the “travelling salesman” problem. The goal of the problem is to find the shortest route between a number of cities, going through each city only once. As you add more cities to the problem, the problem becomes more difficult. Adleman chose to find the shortest route between seven cities. DNA computers have the potential to take computing to new levels, picking up where Moore’s Law leaves off. There are several advantages to using DNA instead of silicon: • As long as there are cellular organisms, there will always be a supply of DNA. • The large supply of DNA makes it a cheap resource. • Unlike the toxic materials used to make traditional microprocessors, DNA biochips can

be made cleanly. • DNA computers are many times smaller than today’s computers. DNA’s key advantage is that it will make computers smaller than any computer that has come before them, while at the same time holding more data. One kg of DNA has the capacity to store more information than all the electronic computers ever built; and the computing power of a teardrop­sized DNA computer, using the DNA logic gates, will be more powerful than the world’s most powerful supercomputer. More than 10 trillion DNA molecules can fit into an area no larger than 1 cubic centimeter (0.06 cubic inches). With this small amount of DNA, a computer would be able to hold 10 terabytes of data, and perform 10 trillion calculations at a time. By adding more DNA, more calculations could be performed. Unlike conventional computers, DNA computers perform calculations parallel to other calculations. Conventional computers operate linearly, taking on tasks one at a time. It is parallel computing that allows DNA to solve complex mathematical problems in hours, whereas it might take electrical computers hundreds of years to complete them. The first DNA computers are unlikely to feature word processing and e­mailing programs. Instead, their powerful computing power will be used by national governments for cracking secret codes, or by airlines wanting to map more efficient routes. Studying DNA computers may also lead us to a better understanding of a more complex computer — the human brain. A DNA computer is a tiny liquid computer —­ DNA in solution — that could conceivably do such things as monitor the blood in vitro. If a chemical imbalance were detected, the DNA computer might synthesize the needed replacement and release it into the blood to restore equilibrium. It might also eliminate unwanted chemicals by disassembling them at the molecular level, or monitor DNA for anomalies. This type of science is referred to as nanoscience, or nanotechnology, and the DNA computer is essentially a nanocomputer.

Discovery of DNA Computer

Class 12 / 37 E d u h e a l F o u n d a tio n

IF YOU HAVE PROBLEMS, DISSOLVE

THEM…..

AS COMPUTER COMPONENTS SHRINK YEAR BY YEAR SCIENTISTS DREAM OF THEIR ULTIMATE GOAL. A CHEMICAL COMPUTER, WHOSE WORKING PARTS WOULD BE INDIVIDUAL MOLECULES.

BUT THIS HAS REMAINED ONLY A DREAM-UNTIL NOW. LEONARD ADLEMAN OF THE UNIVERSITY OF SOUTHERN CALIFORNIA HAS JUST SHOWN HOW TO DO COMPUTATION USING DNA.

ADLEMAN, A COMPUTER SCIENTIST, CHOSE A TASK THAT REPRESENTS A WHOLE CLASS OF HARD-TO-SOLVE PROBLEMS. COMPUTER GUYS CALL IT THE TRAVELING SALESMAN PROBLEM.

COULDN‛T YOU CALL IT

SOMETHING A LITTLE LESS

GENDER BIASED….A LITTLE

MORE NOW?

IN THIS VERSION, THE MARKETING REP* HAS A MAP OF SEVERAL CITIES WITH ONE-WAY STREETS BETWEEN SOME OF THEM. THE PROBLEM IS TO FIND A ROUTE (IF IT EXISTS) THAT PASSES THROUGH EACH CITY EXACTLY ONCE, WITH A DESIGNATED BEGINNING AND END.

TOO CORPORATE! HOW ABOUT THE HAMILTONIAN

PATH PROBLEM?

HOW ABOUT THE MOBILE

MARKETING REP PROBLEM?

WHEN THE NUMBER OF CITIES IS LARGE – SAY MORE THAN 100-THIS

PROBLEM IS TOO MUCH FOR EVEN THE FASTEST

COMPUTER.

* REP = representatvie

Adleman and Discovery of DNA Computer

38 / Class 12 E d u h e a l F o u n d a tio n

HE REPRESENTED EACH CITY CHEMICALLY BY A SINGLE STRAND OF DNA 20 BASES LONG ITS SEQUENCE CHOSEN AT RANDOM.

FOR HIS DNA COMPUTATION, ADLEMAN CHOSE THIS SIMPLE ARRANGEMENT OF 7 CITIES AND 19 STREETS.

THE ACTUAL SEQUENCES

DON‛T MATTER!

A STREET BETWEEN TWO CITIES IS THE COMPLEMENTARY 20 BASE STRAND THAT OVERLAPS EACH CITY‛S STRAND HALFWAY. THIS STREET LITERALLY JOINS THE TWO CITIES.

IN DNA C ALWAYS PAIRS WITH G AND T ALWAYS PAIRS WITH A. SO IN CLOSE-UP IT LOOKS LIKE THIS.

NOTE: SOME CITIES MY BE VISITED MORE

THAN ONCE.

A MULTICITY TOUR BECOMES A PIECE OF DOUBLE-STRANDED DNA, WITH THE CITIES LINKED IN SOME ORDER BY THE STREETS.

ADLEMAN TOSSED A FEW GRAMS OF EVERY DNA CITY AND STREET-WELL OVER 100 TRILLION MOLECULES – INTO A TINY TEST TUBE.

ALL THOSE MOLECULES COMBINED LIKE MAD, MAKING MULTIPLE COPIES OF EVERY POSSIBLE PATH IN AN INSTANT.

OW! GOT DNA IN MY EYE!

Class 12 / 39 E d u h e a l F o u n d a tio n

THE COMPUTATION WAS DONE, BUT WHERE WAS THE ANSWER! SOMEHOW THE PATH THAT VISITS EACH CITY ONCE HAD TO BE EXTRACTED!

Well?

THE LAB WORK TOOK ABOUT A WEEK.

THIS LAB WORK IS

WET ? AND IT

WORKED! IN THE END,

ADLEMAN HAD PURE DNA

THAT ENCODED THIS

7-CITY TOUR!

USING CHEMICAL TECHNIQUES, ADLEMAN FOLLOWED THESE STEPS.

1. EXTRACT ALL PATHS GOING FROM ‘START‛ TO ‘END‛.

2. OF THOSE FIND THE ONES PASSING THROUGH 7 CITIES.

3. OF THOSE ISOLATE PATHS WITH 7 DIFFERENT CITIES.

4. IF THERE‛S ANYTHING LEFT AFTER STEP 3, DECLARE IT THE WINNER.

ONE SECOND TO DO THE COMPUTATION, 600,000 SECONDS TO GET THE OUTPUT!

WHAT DOES IT MEAN, THIS MIRACULOUS MARRIAGE OF COMPUTER SCIENCE AND MOLECULAR BIOLOGY?

THIS IS THE GREATEST THING SINCE SLICED BREAD!!

SLICED BREAD GOES STALE

FASTER THAN UNSLICED

BREAD…

THIS EXPERIMENT WAS ONLY A SMALL BEGINNING AND ALREADY SCIENTISTS ARE EXTENDING THE IDEA TO MORE AND BIGGER PROBLEM.

ANY PROBLEM REQUIRING A BRUTE FORCE

SEARCH OF ALL POSSIBLE

SOLUTIONS.

40 / Class 12 E d u h e a l F o u n d a tio n

ADLEMAN DIVISIONS DNA COMPUTERS WITH TRILLIONS OF PROCESSORS

WORKING IN PARALLEL – SO NOW SOMEONE HAS TO INVENT SOME TRILLION-PROCESSOR SOFTWARE.

And then-

HE NOTES THAT CHEMICAL COMPUTERS WOULD CONSUME ALMOST NO ENERGY OR DESK SPACE.

NOW WHERE IS THAT TEENY-WEENY

KEYBOARD?

ADLEMAN ALSO WORKS TO ENCOURAGE BIOLOGISTS TO

THINK ABOUT DNA PROCESSES IN TERMS OF COMPUTATION.

HOW HAVE COMPUTATIONAL FACTORS ALREADY AFFECTED GENETICS AND EVOLUTION?

WHAT NEW TRICKS CAN WE COAX DNA TO DO?

LEN WAKE UP!

TIME FOR YOUR JOB,

MS. LOMAN.

AND THE EFFECT ON MARKETING MAY BE INCALCULABLE.

Class 12 / 41 E d u h e a l F o u n d a tio n

In the middle of the 19th century, the now­ famous monk Gregor Mendel performed his landmark experiments indicating that certain traits can be inherited, and he postulated a discrete unit of inheritance that we now call a gene. Since then, scientists have come to appreciate how much of an individual’s constitution is determined by genes, and, in particular, they have focussed on the link between genes and disease. Almost all drugs act by altering the phenotype of the target cell, and may therefore be called phenotypic drugs. Among currently marketed drugs, the only exceptions to this rule are certain antineoplastic agents and anthelminthics. When pharmacological agents alter the genetic make­ up of the cell, they may be called genotypic drugs. Almost all gene therapy falls into the category of genotypic pharmacology. Basic requirements for Gene Therapy Gene therapy offers a new treatment paradigm for curing human disease. Rather than altering the disease phenotype by using agents which interact with gene products, or are themselves gene products, gene therapy can theoretically modify specific genes resulting in disease cure following a single administration. Initially gene therapy was envisioned for the treatment of genetic disorders, but is currently being studied in a wide range of disease, including cancer, peripheral vascular diseases, arthritis, neurodegenerative disorders and other acquired diseases.

Gene identification and cloning Even though the range of gene therapy strategies is quite diverse, certain key elements are required for a successful gene therapy strategy. The most elementary of these is that the relevant gene must be identified and cloned. Now upon completion of the Human Genome Project, gene availability will be unlimited.

GENE THERAPY

It has been the hope of biomedical researchers to find ways to fix the genes that cause disease. Over a decade ago, when researchers realized that viruses could be modified to carry corrective genes into cells, gene therapy seemed to be an eventuality. But to date, no one has found a way to reliably control the therapeutic genes to make them clinically useful. The article takes a peek at the future of gene therapy, where the emphasis may be less on replacing defective genes and more on correcting them.

42 / Class 12 E d u h e a l F o u n d a tio n

Gene transfer and expression Once the gene has been identified and cloned, the next consideration must be expression. Questions pertaining to the efficiency of gene transfer and gene expression remain at the forefront of gene therapy research. Currently much debate in the field of gene therapy revolves around the transfer of desired genes to appropriate cells, and then obtaining sufficient levels of expression for disease treatment. Hopefully, future research on gene transfer and tissue­specific gene expression will resolve these issues in the majority of gene therapy protocols. Other important considerations for a gene therapy strategy include : a sufficient understanding of the pathogenesis of the targeted disorder, potential side effects of the gene therapy treatment, and understanding of the target cells to receive the gene therapy.

Unique terminology of Gene Therapy Like most fields, gene therapy has unique terminology. The list provided below will clarify the meaning of some of the common terms. Gene transfer Ex vivo : transfer of genetic material to cells located outside the host. Following transfer of the genetic material, the cell are then implanted back into the host. This term has also been called the indirect method of gene transfer. Gene Transfer In vivo : transfer of genetic material to cells located within the host. This has also been termed the direct method of gene transfer. Gene therapy : the transfer of selected genes into a host with the hope of ameliorating or curing a disease state. Cell therapy (genome therapy) : The transfer of entire cell, that have not been genetically modified, into a host with the hope that the transferred cells with engraft into and improve host function. Somatic gene transfer : Transfer of genes to non­germline tissues in the hope of correcting the disease state of a patient. Germline gene : Transfer of genes to germline (eggs or sperm) tissue in the hope of altering the genome of future generations. Transgene : The selected gene tested in a gene transfer experiment. For example, if you wished to treat a patient for phenylketonuria, you might plan to transfer a corrected version of the phenylalanine hydroxylase gene into the liver cells in this example, the correct version of the phenylalanine hydroxylase gene would be the transgene. Reporter gene : Genes which are used to test the efficiency of gene transfer. Examples include genes encoding luceriferase, galactosidase and chloramphenicol acetyltransferase. Gene transfer vector : the mechanism by which the gene is transferred into a cell. Transfer efficiency : the percentage of cells which are expressing the desired transgene.

Current Status of Gene Therapy Almost daily newspaper reports about the discovery of a new gene that contributes to some disease or another, be it sickle cell disease, muscular dystrophy, familial hyper cholesterolemia, Alzheimer’s or some form of cancer. Right now, therapies directed toward these conditions can only alleviate the symptoms — the manifestations of the defective genes. Implicit, and sometimes explicit, in stories about genetic discoveries is the idea that

Class 12 / 43 E d u h e a l F o u n d a tio n

new therapies can be created that directly address the source of the problem. These gene therapies seek treatments, even cures, that act at the level of the gene itself. Most of the gene therapy techniques developed so far are of the gene­addition variety; that is, they attempt to provide a good copy of a gene to a cell that harbors a bad one. The hope is that the good, corrective gene will compensate for the bad one and restore the cell to its proper function. Gene addition has been achieved by a variety of means — not only in test­tube experiments, but in clinical trials involving real patients as well. Yet, to date, the results of these trials have been disappointing. Even the most successful clinical trial has fallen short of therapeutic efficacy. The majority of the therapy protocols focus on treating acquired diseases such as cancer or HIV. Rest focus on inherited disorders and other disorders like treating peripheral vascular disease, rhematoid arthritis, and arterial restenosis. Cancer protocol strategies Cancer gene therapy protocols employ a wide variety of strategies and can be grouped as follows : In vitro insertion of a cytokine gene into tumor cells; in situ injection of an HLA gene; in situ insertion of a suicide gene into tumor cells; use of tumor suppressor gene or anti­oncogens; and use of the multidrug resistant gene. Gene therapy vectors The majority of protocols employ retroviral vectors to deliver the selected gene to the target cells. Other widely used vectors include adenoviral vectors, liposomes and adenoassociated vectors. Gene Addition Abnormal cell behavior is often the result of an altered gene whose expression is either absent or unregulated. A mutation in just one gene can sometimes cause a cell to malfunction. The mutated gene directs the synthesis of a dysfunctional protein, with the consequence that the cell functions marginally or not at all. In the case of sickle cell anemia, for example, a mutated hemoglobin molecule actually distorts the red blood cell in which it resides, causing the cell to assume a sickle shape instead of its usual disk shape. The shape change prohibits the cell from adequately performing its designated role of carrying oxygen to the body’s organs and tissues. Another example is presented by muscular dystrophy, which is linked to mutations in

chromosomes defective gene

gene addition

nucleus

or

Gene addition seeks to compensate for a defective gene by providing cells with a corrective gene. Genes can be injected directly into cells, or they can be coaxed in by chemical or electrical means. Most delivery systems deposit corrective genes in the cell’s nucleus, where it remains only transiently. Other methods integrate genes into the chromosomes. Integrated genes can be passed on to progeny cells in the course of normal cell division, which may provide long­term therapeutic benefits.

44 / Class 12 E d u h e a l F o u n d a tio n

the dystrophin gene. This gene codes for the dystrophin protein, which is crucial for the strength and movement of normal muscle tissue. People lacking dystrophin experience the muscle weakness characteristic of the disease. Finally some genetic mutations do not alter a cell’s function as much as they interfere with the cell’s normal life cycle, specifically its cell­division cycle. Such mutations can lead the cell to divide uncontrollably, as is the case in certain cancers. The essence of gene therapy, then, is to deliver to a cell a correct version of a mutated gene, the expression of which will produce the normal protein and hence restore normal cellular function. This has been obvious for some time, but how to achieve this goal has not been. An initial problem centered on how to get a gene into a cell. The chromosomes of a mammalian cell are housed inside a membrane­bounded compartment, called a nucleus. It is not enough for a gene­delivery system to deposit the gene into the cell; the gene must be delivered to the nucleus.

This in itself is not difficult. Scientists have been able to do it for decades. Foreign DNA can be injected into a cell, or its entry can be facilitated by various chemical or electronic means. But these methods are not very efficient, and one requirement for gene therapy is that sufficient amounts of corrective DNA be delivered to enough cells to be therapeutically beneficial. Under the best c i rcums tances , one would also want the therapeutic DNA to become a permanent part of the host’s c h r omo some s . This would ensure its stabili ty and would mean that the therapeutic gene would be replicated along with the host’s c h r o m o s o m e s during each cell division. In contrast, DNA delivered to a cell

virus gene

virus

corrective gene

viral vector

Viruses can be used as gene­delivery vectors. Viral genes targeting the cell’s nucleus are retained in the vector, while harmful viral genes are removed and replaced with the corrective gene. Some viruses integrate corrective genes into chromosomes, but randomly.

Receptor

Viral vector

(a) (b)

(c) (d)

Natural ability of viruses to enter cells through recep­ tors can be exploited for gene therapy. Viruses recog­ nize and attach to receptors (a) and work their way through, into the cell (b). Once inside, the virus dis­ charges its contents (c). Viral genes progress through the cell and into the nucleus (d). Depending on the specific virus, these genes may or may not integrate into the host’s chromosomes. When the virus has been modified to function as a vector, the genes it transports into the cell are corrective genes. Each virus is particu­ larly adapted to use one or a few specific receptors, which limits the range of cells each one can infect.

Class 12 / 45 E d u h e a l F o u n d a tio n

by physical or chemical means can be placed in the cell’s nucleus and can be expressed, but it does not become integrated into the chromosomes. An ideal gene­delivery vehicle would be able to enter a large number of cells and integrate its DNA into the host’s chromosomes. As it happens, some kinds of viruses are perfectly adapted to do just that. And, about 15 years ago, Richard Mulligan and Constance Cepko, who were then at Massachusetts Institute of Technology, along with colleagues at MIT and Harvard, made the important technological leap that initiated the modern era of gene therapy. Specifically they demonstrated that members of the retrovirus family could be engineered to carry foreign genes into mammalian cells and splice them into the host’s chromosomes. To create these gene­delivery vectors, Mulligan and his coworkers essentially gutted the virus of its genes, disposing of those that could be harmful to the host. At the same time, they retained those genes that enable the retrovirus to insert DNA into host chromosomes. By attaching this integrative machinery to the therapeutic gene, they created a retrovirus capable of infecting cells and splicing a corrective gene into chromosomes. Inserting a gene, however, is only half of the problem. The vector must also contain a mechanism for activating the therapeutic gene, since this is not automatic. Genes have evolved a pattern of expression wherein certain levels of their product are required at specific times in the life cycle of the cell. Hence the corrective action of gene therapy must include a timing and regulatory “device.” Such devices are usually found at the start of a gene and constitute the gene’s “on” switch, or promoter. But this leads to another problem. Promoters are often exquisitely complex and sometimes quite large, so placing them into a therapeutic vector is difficult. When constructing their retroviral vectors, Mulligan and his colleagues opted to use promoters native to the virus, rather than the corrective gene’s own promoter. In laboratory petri dishes, these vectors sometimes worked quite well, but not always. In some cases, the therapeutic genes entered the cells as expected but were expressed at unpredictably low levels. Low levels of expression continue to dog gene­ therapy efforts, and improving expression

vector

retrovirus

adenovirus

adeno­associated virus

herpesvirus

advantages •enters cells efficiently •viral genes absent •integrates stably

•enters cells efficiently •produces high expression of therapeutic gene

•does not integrate into host chromosome

•Integrates into chromosome at specific site

•does not produce immune response

•produced at high levels •targets nondividing nerve cells

disadvantages •hard to produce •limited insert size •random mutagenesis

•viral genes must be in vector

•induces immune response

•small insert size allowed

•hard to produce

•hard to produce •viral gene required

Viruses vary in their usefulness as gene­therapy vectors. Some viruses are more adept at getting into cells, whereas others may ensure that corrective genes are expressed at higher levels or for longer periods. No one vector seems to combine all of the desirable properties.

46 / Class 12 E d u h e a l F o u n d a tio n

levels remains a major focus of research. Recent vectors include portions of the gene’s own promoter. This has the added benefit that the therapeutic gene is expressed as naturally as possible — only during the times when its product is needed. Other constructions attach promoters that can be externally controlled. For example, certain genes have promoters that are sensitive to the antibiotic tetracycline and are activated when the drug is present. A vector was recently constructed by Herman Bujold and colleagues at the University of Heidelberg that pairs a tetra cycline­sensitive promoter with a corrective test gene. The test gene would be activated only if the patient ingests tetracycline. The initial expectation was that cells would have to be removed from the body in order to be treated. This ex vivo approach would necessarily limit therapy to those cells, such as blood cells, that are easily removed and replaced. But more recently, retroviral vectors have been developed that can be infused directly into an organ, such as the liver, or placed into the lung by inhalation. This versatility is one of the great advantages of retroviral vectors. There are also some considerable disadvantages to retroviral vectors that have made investigators cautious about using them. The same feature that makes the retroviruses so attractive to gene­therapy investigators has also been one of their greatest drawbacks — namely the ability to integrate genes into chromosomes. The problem is that scientists have no control over how many copies of the gene become integrated or where on the chromosome they insert. Since integration appears to be essentially random, the vector’s genetic payload may become inserted within another important gene, disrupting or altering its expression. Or a gene may integrate within the regulatory region of a gene responsible for controlling cellular proliferation, thus putting the cell on the path towards cancerous growth. Although these are remote possibilities, they are real and must nevertheless be considered as a potential consequence of retroviral­based gene­delivery vectors. Adenovirus and Others : One of the most promising vehicles to emerge from recent gene­therapy studies is adeno­associated virus (AAV). This virus infects a wide range of cells, including lung and muscle cells, and it integrates its genes within the host’s. In addition, it can infect nondividing cells and does not elicit an immune response — both of which are important advantages over retroviral and adenoviral vectors. The work on this virus has been pioneered by three investigators: Kenneth Berns and Nicholas Muzyczka at the University of Florida and R. Jude Samulski at the University of North Carolina at Chapel Hill. Significant advances in the use of AAV for gene therapy have recently been reported by Mark Kay and colleagues at Stanford University and Kathryn High and coworkers at the University of Pennsylvania. Both of these research groups used a modified AAV vector to achieve long­term expression and correction in animals of a gene that contributes to hemophilia. This achievement required a detailed appreciation for the basic biology of AAV. However, as expected, this virus has some drawbacks. First, it can carry only a small genetic payload, which considerably restricts its usefulness. Second, it, too, carries the risk of disrupting functioning genes by randomly inserting itself into the chromosomes.

Class 12 / 47 E d u h e a l F o u n d a tio n

Finally, it is somewhat difficult to manufacture these vectors in sufficiently high quantities. Other viruses under study as potential vector candidates include Herpes simplex, Vaccinia and even the human immunodeficiency virus. In addition to viral­based vectors, investigators are continuing to explore nonviral delivery systems. One system that holds some promise delivers drugs via liposomes, small vesicles artificially created from lipids that resemble those making up the membranes of mammalian cells. Because they are constructed of virtually identical materials, the liposomes can fuse with cell membranes and empty their contents — which can include drugs or corrective genes — inside the cell. Some of the DNA delivered by liposomes makes its way into the cell’s nucleus. Targeted Gene Repair Ultimately, scientists would like to replace a dysfunctional gene with a functional one, within the normal context of the chromosome, an approach that could skirt the concerns about the number of genes delivered, the chromosomal location and the level of expression. Right now, homologous recombination, the only technique that comes close to this, is so inefficient that its success rate is 1 in 10,000. Needless to say, this is not adequate for human use. But the idea of completely replacing a bad gene with a good one may be overreaching, especially when one considers how small are many of the mutations that contribute to disease. To understand how small, we must first consider a few basic facts about the composition of genes. The gene is to inheritance is what a word is to language; it is the basic unit of meaning. In the genetic lexicon, the gene is a length of DNA that codes for a particular protein. The alphabet used by the genetic language contains only five letters, or nucleotides, named for the bases. These are adenine (A), thymine (T), cytosine (C), guanine (G) and uracil (U). The nucleotides A, T, C and G are found in DNA. The average human gene is a l ittle over 1,000 nucleotides long. In many inherited disorders only one or a few of these nucleotides is incorrect. For example, sickle cell anemia is the result of a single nucleotide substitution, a single letter misspelled, in the gene encoding the b­globin strand of hemoglobin. Yet this one­nucleotide substitution can cause the structural deformity of the molecule and the characteristically distorted shape of the sickled red blood cell. Over 70 percent of the cases of cystic fibrosis are attributable to the deletion of three nucleotides in the CFTR gene. Why should the entire gene be replaced when the error is so minimal? That strategy seems akin to

gene replacement

Gene replacement is theoretically more desirable than gene addition. By replacing the defective gene with the corrective one at the exact site on the chromosome, where the normal gene is supposed to be, scientists hope to achieve natural gene regulation — the corrective gene is expressed at the times and in the amounts that the natural gene would be. The techniques currently available to do this are so inefficient that gene replacement is not yet practical.

48 / Class 12 E d u h e a l F o u n d a tio n

remodeling the whole kitchen to repair a leaky faucet. In 1993, while studying homologous recombination in mammalian cells, scientists began experimenting with ways to repair damaged genes, rather than replacing them. The cell’s own repair mechanisms are extremely efficient, as evidenced by the simple and continual inheritance of normal genes through generations of cell divisions. If could be harnessed the cell’s own power of DNA repair they might be able to correct mutations. Normal human chromosomes are actually made up of two strands of DNA complexed to each other in an interesting way. It turns out that the nucleotides of DNA can bind with each other in a specific pattern. Except in very rare cases, adenine always pairs with thymine, and guanine always pairs with cytosine. Each DNA strand carries a nucleotide sequence exactly complementary to the other, such that every adenine nucleotide on one strand is matched up with a thymine on the partner strand, and every guanine is matched with a cytosine on the complementary strand. A sequence of GATC on one strand would therefore bind to the sequence of CTAG on its partner, or so it should be. Occasionally the wrong nucleotide is inserted into a spot, so that the corresponding nucleotide on the partner strand cannot properly bind in that position. In that case the mismatched nucleotides form a bulge. Usually this is not a problem, since the cell contains DNA repair mechanisms that actually scan the DNA and detect such bulges. When one is discovered, the repair systems work to remove the incorrect nucleotide and replace it with the correct one. But if the mismatch is overlooked by the cell’s repair machinery, the

Gene correction exploits cellular proofreading enzymes that detect errors in DNA and make corrections. Scientists place into the cell a small hybrid RNA­DNA molecule called a chimeric oligomer that pairs with the defective gene in the region of the error (a). Repair enzymes use the oligomer as a template to guide the correction. Seen close up (b), the oligomer binds snugly with the defective gene except in the region of the error, where the mismatch causes a bulge. Repair enzymes detect this bulge and replace the erroneous nucleotides. In this example the guanine (G)­cytosine (C) pair is incorrect. The oligomer provides the template indicating that an adenine (A)­thymine (T) pair should be inserted in that spot. The repair enzymes follow the instructions in the template and correct the gene accordingly. Corrections made this way endure for generations of cell divisions.

incorrect gene

enzyme

chimeric oligomer

correct segment G

corrected gene A

T

A

C

G

C chromosome

enzyme

correct segment

RNA

chimeric oligometer

incorrect segment

enzyme (a) (b)

correct segment

Class 12 / 49 E d u h e a l F o u n d a tio n

error is retained, and the gene remains defective. To alert these repair mechanisms to the error. Repair enzymes remove the erroneous nucleotide and replace it with a nucleotide complementary to the one in that position in the oligomer, which happens to be the correct nucleotide.

50 / Class 12 E d u h e a l F o u n d a tio n

DNA FINGERPRINTING ANALYSIS How does forensic identification work? Any type of organism can be identified by examination of DNA sequences unique to that species. Identifying individuals within a species is less precise at this time, although when DNA sequencing technologies progress farther, direct comparison of very large DNA segments, and possibly even whole genomes, will become feasible and practical and will allow precise individual identification. To identify individuals, forensic scientists scan 13 DNA regions that vary from person to person and use the data to create a DNA profile of that individual (sometimes called a DNA fingerprint). There is an extremely small chance that another person has the same DNA profile for a particular set of regions. Some Examples of DNA Uses for Forensic Identification • Identify potential suspects whose DNA may match evidence left at crime scenes. • Exonerate persons wrongly accused of crimes. • Identify crime and catastrophe victims. • Establish paternity and other family relationships. • Identify endangered and protected species as an aid to wildlife officials (could be used

for prosecuting poachers). • Detect bacteria and other organisms that may pollute air, water, soil, and food. • Match organ donors with recipients in transplant programs. • Determine pedigree for seed or livestock breeds. • Authenticate consumables such as wine. Is DNA effective in identifying persons? DNA identification can be quite effective if used intelligently. Portions of the DNA sequence that vary the most among humans must be used; also, portions must be large enough to overcome the fact that human mating is not absolutely random. Consider the scenario of a crime scene investigation . . . Assume that type O blood is found at the crime scene. Type O occurs in about 45% of world population. If investigators type only for ABO, then finding that the “suspect” in a crime is type O really doesn’t reveal very much. If, in addition to being type O, the suspect is a blond, and blond hair is found at the crime scene, then you now have two bits of evidence to suggest who really did it. However, there are a lot of Type O blonds out there. In this way, by accumulating bits of linking evidence in a chain, where each bit by itself isn’t very strong but the set of all of them together is very strong, you can argue that your suspect really is the right person. With DNA, the same kind of thinking is used; you can look for matches (based on sequence or on numbers of small repeating units of DNA sequence) at a number of different locations on the person’s genome; one or two (even three) aren’t enough to be confident that the

Class 12 / 51 E d u h e a l F o u n d a tio n

suspect is the right one, but four (sometimes five) are used and a match at all five is rare enough that you (or a prosecutor or a jury) can be very confident (“beyond a reasonable doubt”) that the right person is accused. DNA fingerprinting is a laboratory procedure that requires six steps : 1) Isolation of DNA. DNA must be recovered from the

cells of the plant or animal. Only a small amount is needed. For example, the amount of DNA found in one drop of blood or one square centimeter (about the size of a dime) of leaf tissue is usually sufficient.

2) Cutting, sizing, and sorting the DNA. Special enzymes called restriction enzymes are used to cut the DNA at specific places. For example, an enzyme called EcoR1 found in bacteria will cut DNA only when the sequence GAATTC occurs. The DNA pieces are sorted according to size by a sieving technique called electrophoresis through a gel made from seaweed agarose. Electrophoresis is the DNA equivalent of sieving sand through progressively finer mesh screens to determine particle size distribution.

3) Transfer of DNA to nylon. The DNA pieces are transferred to a nylon sheet by placing the agarose gel and nylon next to each other overnight.

4­5)Probing. The DNA fingerprint is generated by adding tagged probes to the nylon sheet. Each probe typically sticks in only one or two specific places, wherever the sequences match (A with T and G with C). The tag allows detection of the probe.

6) DNA fingerprint. The final DNA fingerprint is built from several different probes and resembles the bar codes used at the grocery counter.

What are some of the DNA technologies used in forensic investigations? Restriction Fragment Length Polymorphism (RFLP) RFLP is a technique for analyzing the variable lengths of DNA fragments that result from digesting a DNA sample with a special kind of enzyme. This enzyme, a restriction endonuclease, cuts DNA at a specific sequence pattern know as a restriction endonuclease recognition site. The presence or absence of certain recognition sites in a DNA sample generates variable lengths of DNA fragments, which are separated using gel electrophoresis. They are then hybridized with DNA probes that bind to a complementary DNA sequence in the sample. RFLP is one of the original applications of DNA analysis to forensic investigation. With the development of newer, more efficient DNA­analysis techniques, RFLP is not used as much as it once was because it requires relatively large amounts of DNA. In addition, samples degraded by environmental factors, such as dirt or mold, do not work well with RFLP.

1. The process begins with a blood of cell sample from which the DNA is extracted.

2. The DNA is cut into fragments using a restriction enzyme. The fragments are then separated into bands by electrophoresis through an aga rose gel.

3. The DNA band pattern is transferred to a nylon membrane.

4. A radioactive DNA probe is introduced. The DNA probe binds to specific DNA sequences on the nylon membrane.

5. The excess probe material is washed away leaving the unique DNA band pattern.

6. The radioactive DNA pattern is transferred to X­ray film by direct exposure. When developed, the resultant visible pattern is the DNA fingerprint.

The process of DNA fingerprinting

52 / Class 12 E d u h e a l F o u n d a tio n

PCR Analysis PCR (polymerase chain reaction) is used to make millions of exact copies of DNA from a biological sample. DNA amplification with PCR allows DNA analysis on biological samples as small as a few skin cells. With RFLP, DNA samples would have to be about the size of a quarter. The ability of PCR to amplify such tiny quantities of DNA enables even highly degraded samples to be analyzed. Great care, however, must be taken to prevent contamination with other biological materials during the identifying, collecting, and preserving of a sample. Invented in the 1980s by Kary B mullis, it enables us to produce large

Step ­ 4 The mixture is heated to 75°C for at least a minute. This is the optimum temperature for the DNA polymerase enzyme which adds bases to the primer segments. The DNA poly­ merase builds up complementary strands to give two complete DNA molecules identical to the original strand.

Mixture of reactants including the DNA to be amplified, DNA polymerase, the four

nucleotides bases A,T,C and G and primers.

Step 1 A PCR vial containing all the reactants needed to produce millions of identical DNA molecules is placed in a PCR machine. The machine raises and lowers the temperature of the reacting mixture to control the different stages of the reaction.

Steps 2 ­ 4 are repeated 30 times to give around 1 billion copies of the original DNA in just a few hours

Step 2 The reaction mixture is heated to 90­95°C for about 30 seconds. At this temperature the DNA strands separate.

Primers

Original DNA DNA Polymerase

Step 3 The reactants are cooled down to 50­60°C for about 20 seconds. At this temperature the primers, which are short sequences of nucleotide bases, bind to the single DNA

strands.

Class 12 / 53 E d u h e a l F o u n d a tio n

Polymerase chain reaction New horizons in medicine

54 / Class 12 E d u h e a l F o u n d a tio n

quantities of DNA from very small samples in a remarkable short time. This in turn makes it possible for us to analyse tiny samples of DNA and unravel the mysteries of the individual genes. STR Analysis Short tandem repeat (STR) technology is used to evaluate specific regions (loci) within nuclear DNA. Variability in STR regions can be used to distinguish one DNA profile from another. The odds that two individuals will have the same 13­loci DNA profile is about one in one billion. Mitochondrial DNA Analysis Mitochondrial DNA analysis (mtDNA) can be used to examine the DNA from samples that cannot be analyzed by RFLP or STR. Nuclear DNA must be extracted from samples for use in RFLP, PCR, and STR; however, mtDNA analysis uses DNA extracted from another cellular organelle called a mitochondrion. While older biological samples that lack nucleated cellular material, such as hair, bones, and teeth, cannot be analyzed with STR and RFLP, they can be analyzed with mtDNA. In the investigation of cases that have gone unsolved for many years, mtDNA is extremely valuable. All mothers have the same mitochondrial DNA as their daughters. This is because the mitochondria of each new embryo comes from the mother’s egg cell. The father’s sperm contributes only nuclear DNA. Comparing the mtDNA profile of unidentified remains with the profile of a potential maternal relative can be an important technique in missing person investigations. Y­Chromosome Analysis The Y chromosome is passed directly from father to son, so the analysis of genetic markers on the Y chromosome is especially useful for tracing relationships among males or for analyzing biological evidence involving multiple male contributors.

Infection Detection Amplifying the DNA from a single bacterium or virus using PCR can provide a speedy and accurate diagnosis for serious infections, where getting the right treatment quickly can mean the difference between life and death. PCR is already used in the diagnosis of AIDS, viral meningitis, TB and an ever­growing number of other infections.

Genetic Testing PCR makes it easier to identify individuals who carry the genes which can cause problems like cystic fibrosis and muscular dystrophy. In the future it may be used to develop tests for the genetic variations which give an increased risk of heart disease or cancer, and so help everyone to plan a healthy lifestyle.

Cancer Warning Using PCR to amplify the DNA, scientists are developing tests to pick up the genetic changes which take place in cancerous cells very early in the development of the disease. PCR has already made it possible to detect bowel cancer from the DNA of cells extracted from the faeces ­ an easy, quick and non­intrusive way of making a diagnosis which gives the treament a much better chance of success,

Tissue Matching In organ transplants, a close tissue match between the donor and the recipient reduces the chances that the new organ will be rejected. PCR technology is leading to increasingly sophisticated levels of tissue matching at the DNA level and more successful transplants.

Forensic Medicine The ability to amplify the tiniest fragment of DNA found at a crime scene, even years after the event, has resulted in amazing developments in identifying and eliminating suspects. In crimes ranging from murder and rape to theft, PCR, along with DNA fingerprinting, has provided a major breakthrough for the police and forensic teams in the fight against crime.

Class 12 / 55 E d u h e a l F o u n d a tio n

Activity : 1. The data below shows the results of electrophoresis of PCR fragments amplified using probes for the

site which has been shown to be altered in Huntington’s disease. The male parent, as shown by the black box, got Huntington’s disease when he was 40 years old. His children include 6 (3,5,7,8,10,11) with Huntington’s disease, and the age at which the symptoms first began is shown by the number above the band from the PCR fragment. What is the prognosis for the normal children 4, 6, and 9?

A. 4 and 9 do not have the trait, and will not get Huntington’s disease, but 6 is likely to start the disease when he reaches his father’s age of 40. B. 4, 6, and 9 are lucky and have not inherited

the defect causing Huntingtington’s disease. C. 4, 6, and 9 will still develop Huntington’s

disease at some point in their lives, since the disease is inherited as a dominant trait.

D. Two of the three will develop the disease, since it is inherited as a dominant trait, but the data does not allow you to predict which two.

E. 4, 6, and 9 must be children of a different father, and thus do not carry the trait for Huntington’s disease.

2. The data below shows the results of electrophoresis of PCR fragments amplified using probes for the site which has been shown to be altered in Huntington’s disease. The male parent, as shown by the black box, got Huntington’s disease when he was 40 years old. His children include 6 (3,5,7,8,10,11) with Huntington’s disease, and the age at which the symptoms first began is shown by the number above the band from the PCR fragment. In the Figure showing data on Huntington’s disease, which of the following conclusions is valid: A. No relationship between age of onset of disease and the migration rate of PCR fragments. B. A shorter PCR fragment predicts early onset of Huntington’s disease. C. Increased length of the amplified PCR fragment predicts early onset of Huntington’s disease. D. Huntington’s disease must be contagious since many of the children have the disease. E. None of the above.

3. “Gene library” is a term used to describe: A. a computerized listing of known DNA sequences.

1 2 3 4 5 6 7 8 9 10 11

100 2

14

11

27 26

40 3H

1 2 3 4 5 6 7 8 9 10 11

100 2

14

11

27 26

40 3H

56 / Class 12 E d u h e a l F o u n d a tio n

B. bacteria with plasmids containing DNA fragments representing the majority of the genetic information from a plant or animal.

C. a collection of books about recombinant DNA technology. D. a compilation of the amino acid sequences of protein coding genes. E. a store that specializes in the sale of Levis.

4. One of the most significant discoveries which allowed the development of recombinant DNA technology was: A. the discovery of antibiotics used for selecting transformed bacteria. B. the identification and isolation of restriction endonucleases permitting specific DNA cutting. C. the discovery of DNA and RNA polymerase allowing workers to synthesize any DNA

sequence. D. the development of the polymerase chain reaction. E. the Southern technique for separation and identification of DNA sequences.

5. A key feature of insertional mutagenesis for the identification of plasmids containing recombinant DNA is: A. the production of nutritional auxotrophs. B. the DNA sequencing of recombinant plasmids. C. the production of restriction endonuclease maps of recombinant plasmids. D. introns can be moved to new locations within the gene. E. the disruption of a gene on the plasmid by the inserted recombinant DNA.

ANSWERAND EXPLANATION TOACTIVITIES

1. During electrophoresis, fragments of DNA migrate through a gel with larger fragments moving more slowly. In Huntington’s disease, PCR amplifies a region of the chromosome which has variable number of repeating CAG sequences. Normal individuals can have up to 30 copies of the sequence but individuals with Huntington’s have from 37 to over a hundred. In the gel showing results of the PCR experiment for a family with a father having Huntington’s disease, children 4 and 9 show only normal sizes for this region, showing that they will not get the disease. The normal individual 6 has amplified sequences similar to the diseased father, showing that he too will get Huntington’s disease. The size of the CAG fragment correlates with the age of onset. Since the father got the disease at age 40, and child 6 has the same size of the fragment, we can suggest that he is likely to get Huntington’s disease about the time he reaches 40. Ans. (A) : 4 and 9 do not have the trait, and will not get Huntingtons disease, but 6 is likely to start the disease when he reaches his father’s age of 40. Children 4 and 9 do not have an amplified CAG repeat, and their PCR product migrates with control normals. Child 6 is currently normal, but has an allele with approximately the same number of repeats as the father. Thus, you could expect the child to develop Huntington’s disease when he/she reaches 40.

2. Relationship between the number of CAG repeats and the age of onset of the disease

Class 12 / 57 E d u h e a l F o u n d a tio n

The PCR amplified region shows a correlation between size of the fragment, a measure of the number of times CAG is repeated, and the age of onset. Larger fragment size correlates with earlier disease. Ans (C) : Increased length of the amplified PCR fragment predicts early onset of Huntington’s disease. There is an inverse relationship between the number of CAG repeats and the age at which Huntington’s disease begins.

3. When the genomic DNA is digested by a restriction endonuclease, and all fragments cloned at random into a plasmid vector, then the majority of genetic information will be included in the mixture of bacteria. Cultures of the bacteria, with each containing only a fraction of the genome, collectively contain all the genes and are called a library. Ans (B) : bacteria with plasmids containing DNA fragments representing the majority of the genetic information from a plant or animal. A library is a culture of bacteria where each cell has one or a few DNA sequences from another organism, but the whole culture contains the majority of the DNA fragments from an organism.

4. Restriction endonucleases Restriction endonucleases — cut double stranded DNA at specific sequences, protection against viruses in bacteria.

Sequences may be palindromes: a sequence which is the same when read in either direction. “Able was I ere I saw Elba”

Example of specificity of restriction endonucleases

Name Source Microorganism Recognition Sequence

Bam HI Bacillus amyloliquefaciens G↓GATCC Eco RI Eschericia coli RY 13 G↓AATTC Hind III Haemophillus A↓AGCTT

influenzae Rd NoI I Nocardia otitidis­caviarum GC↓GGCCG PsI I Providencia stuartil CTGCA↓G Sma I Serratia marcescens CCC↓GGG

Restriction endonucleases allow the specific and reproducible fragmentation of DNA. The discovery of these enzymes allowed the development of modern recombinant DNA technology.

restriction endonuclease protected

(methylene d) recognition sites

Bacterial chromosome

incoming phage DNA (unprotected) cleaved by restriction endonuclease

58 / Class 12 E d u h e a l F o u n d a tio n

Ans (B) : the identification and isolation of restriction endonucleases permitting specific DNA cutting. Prior to the discovery of these enzymes, there was no reproducible way to fragment DNA.

5. The plasmid vector contains another identifiable gene (e.g., a second drug resistance or an enzyme activity), with the coding sequence of this gene containing the restriction site for insertion. Insertion of the foreign DNA at this site interrupts the reading frame of the gene and result in insertional mutagenesis.

Example

In this example, the b­galactosidase gene is inactivated. The substrate “X­gal” turns blue if the gene is intact, ie. makes active enzyme. White colonies in X­gal imply the presence of recombinant DNA in the plasmid. Ans. (E) : the disruption of a gene on the plasmid by the inserted recombinant DNA. For example, b­galactosidase on the plasmid can be disrupted by the inserted gene.

cell +pUC19 cell + pUC19 + Insert

Non­transformed cell

Nutrient medium + ampicillin + X­gal

Class 12 / 59 E d u h e a l F o u n d a tio n

Once you get past kindergarten, the alphabet is pretty much old hat. Yet we use this little set of symbols to express lifetimes of thoughts, jokes, dreams, and joys. Living things have a language, too, coded in the order of the nucleotide letters A, C, T, and G in their genes. One of the great scientific quests of the past­that’s the 20th­century was to understand this “language of life.” First, scientists learned that cells store their instructions for living in their DNA. Then, researchers figured out how cells convert the nucleotide sequences in DNA into the sequences of amino acids that make up proteins. Now, scientists are busy reading out complete DNA sequences for whole organisms. They already have a rough draft of virtually the entire human genome, all 3 billion nucleotides of it. Unfortunately, a genome’s worth of raw sequence data is about as comprehensible as a shredded encyclopedia. You might pick out individual words, or even a few paragraphs, but you still can’t readily understand how the whole thing fits together. In the 1990s, scientists developed a new tool for deciphering DNA called a “DNA chip”, also known as a “DNA microarray”. It allows one scientist to collect more information about DNA sequences in an afternoon than an army of scientists could collect in several years using earlier techniques. DNA chips promise to carry the science of understanding genomes to a whole new level, and to bring tools for getting DNA­sequence information out of research labs into doctors’ offices, the better to tailor­ fit medical treatments to an individual’s particular genetic makeup. The brain has about a trillion neurons, and about a quadrillion interconnections. What we call consciousness somehow emerges from how all these neurons interact. We could study an individual neuron for 50 years, and that wouldn’t tell us one bit more about the brain’s emergent properties, because they arise from the network, not a single cell. If we were to study each gene in isolation, we’d never know how the genome functions as a whole. DNA chips are the prototype global technology for genetics, because they let us look at the behavior of thousands of genes at once.

DNA CHIPS A laboratory in the palm of your hand

A hand­held DNA Chip device, made by Nanogen, Inc. The circles at the top are sample ports. The wires guide electric fields over the DNA array, located on the diamond in the centre. It allows one scientist to collect more information about DNA sequences in an afternoon than an army of scientists could collect in several years using earlier techniques.

60 / Class 12 E d u h e a l F o u n d a tio n

How Chips Work DNA chips come in many varieties. Some are “homemade” in scientists’ laboratories, with glass microscope slides and a robot arm wielding a high­tech fountain pen. Private companies are developing other techniques for mass production. But DNA chips all depend on the same basic principle: complementary DNA stands stick together. First, recall that a double­stranded DNA molecule can unzip into two complementary strands. Each of these can zip back together with its complementary sequence. That could be either its old partner, or a new partner with the same sequence. The trick that makes DNA chips work is that you can tether a “new partner” to a flat surface. Imagine a standard checkerboard, 8 squares on a side, 64 squares total. In each square, you tie down a different snippet of single­stranded DNA just three nucleotides long. You write down the sequence in each square. (You can make 64 different sequence variations from three nucleotides­ACG, CGT, GTA, TAC, AAA, and so on­ so there’s just enough room for all the possibilities.) Now imagine you have an unknown sequence, also three nucleotides long. To find out what this unknown is, set it loose on the array so that it wanders from square to square. When your unknown sequence finds its complement, it sticks. To figure out your unknown sequence, all you have to do is find which square your unknown DNA stuck to. Because you know the sequence of the DNA you tied down to that square, you know that the unknown sequence is the complement.

Using Chips What gives DNA chips their power in the real world is their flexibility, compact size, speed, and low cost. Scientists can put not just a hundred but hundreds of thousands of distinct DNA sequences on a microscopic grid a few centimeters across. Then, using fluorescent molecular tags that light up when a complementary strand binds to a particular spot, a person (or a robot) can read out which sequences on the chip find their complement in an unknown sample. DNA chips can gather an incredible variety of data very quickly. And because chips can be mass­produced, they will likely be very inexpensive in the near future. That will allow easy collection of genetic information from many, many individuals, opening up all kinds of opportunities to help doctors diagnose and treat their patients.

Expression Analysis One way DNA chips allow scientists to observe genes working together is called “expression analysis.” (Remember that to “express” a gene as a protein, cells first transcribe the gene’s DNA sequence into a complementary mRNA copy. Then a ribosome translates the

Class 12 / 61 E d u h e a l F o u n d a tio n

Follow through this set of diagrams to understand how chips work. Cellular DNA is double stranded. Chromosomes in a cell’s nucleus contain double stranded DNA. The two strands are complementary—A is opposite T, C is opposite G. Using a single probe.In the 1970’s, scientists learned to use DNA probes to find specific target sequences in solution. First, radioactively label a known DNA sequence, then put it into a mix of unknown sequences. If the probe’s complement is there, it will bind. Look for the label.Next they separated the double­stranded DNA from the single­stranded. If the probe found it’s target, the radioactive label would be in the double stranded fraction. DNA Chips: Thousands of Probes at Once. DNA chips allow scientist to use thousands of probes all at once. First, spot the different probes on a surface, noting sequence they put at each spot. Let The Targets Loose.This time, label the targets in solution and put the solution on the chip. Any targets that find their complementary probes will stick to the surface. Look For The Label. Next, gently wash the surface, and look for the labeled spots. Because you know the sequences of all the probes, you can easily deduce the sequences present in the solution.

62 / Class 12 E d u h e a l F o u n d a tio n

mRNA sequence into the string of amino acids that makes up the protein. Cells constantly switch genes on or off as conditions change. To understand a cell’s behavior in response to a stimulus­the presence of a hormone, or a toxin, or some environmental signal­it would be handy to have a minute­to­minute reading of which genes are turned on. DNA chips are just about perfect for tracking this kind of minute­to­minute change in gene expression. For example, if you wanted to find out the details of how yeast cells make spores. (Other scientists had already determined the DNA sequence of every possible mRNA a yeast cell makes). So, you put the complements of each of these possible mRNA sequences onto a chip. Then, you ground up a bunch of resting yeast cells, which of course contained an mRNA corresponding to each gene that was active at the moment the cells hit the blender. Next, you spread this mixture over the surface of the chip. Only the spots corresponding to genes that were actively churning out their mRNA lit up, because these were the only spots on the chip that had found their complementary sequence. This first experiment gives you a baseline. Next, you stimulate the yeast to form spores (by taking away their food) and repeated the chip analysis six times over the next 12 hours. By looking at which genes turned on, and when, you get many new insights into how yeast cells genetically shift gears to make spores. But the significance of the above experiment you have just performed goes way beyond yeast physiology­it paved the way for using DNA chips to see how dozens of genes work in concert to change a cell’s behavior. Expression analysis has medical applications, too. For example, using expression analysis­made possible by a DNA chip­to develop a test to classify different types of leukemia. (To choose the best treatment, doctors need to know exactly what type of cancer a patient has.). To do this, the researchers looked at samples from about 50 patients already known to have one of two different kinds of leukemia. Then, using the patterns of gene expression they found in the two groups, they correctly predicted which type of leukemia several patients had. In the near future, doctors may be able to use this test to decide which is the best treatment for a new leukemia patient. Researchers also plan to develop similar tests to match treatments to patients for other kinds of cancer, too.

Mapping Our Differences Pick any two people in the world, and you would find their DNA is 99.9 percent identical. The remaining 0.1 percent is the genetic basis of all of humanity’s differences, from the shape of our faces to the way some people get cancer to the fact that some patients respond to a certain drug while others don’t. Scientists are now starting to use DNA chips to map out tiny one­letter variations in the 3­billion­nucleotide human genome. These pinpoint differences are called “single nucleotide polymorphisms,” or SNPs. Identifying them will help researchers to understand the basis for human variation.

Class 12 / 63 E d u h e a l F o u n d a tio n

But to map SNPs, you need a different kind of chip. For expression analysis, you use a chip containing all possible genes. For SNP work, you make a chip with many, many possible variations of one gene. Then you take a DNA sample from the person you want to test, use PCR to make multiple copies of the gene you’re interested in, and put this “amplified” sample on the chip. The spot that lights up will correspond to the particular sequence variant the person has. Because the test is quick and not too expensive, you can do many of them. Then, you correlate different outcomes­response to a certain drug, for example, or the probability of getting heart disease­with the different genetic variations. There are only about 200,000 functionally important variants [SNPs] in the human genome that have reasonable frequencies. Nearly all of the genetic contributions to diabetes and heart disease and hypertension and all of the common illnesses are found in those 200,000 elements. Once researchers know which SNPs correlate with higher risk for disease, people with these traits will be able to take extra steps to avoid getting sick. This might allow medicine to move from its present mode, where we spend most of our resources treating people who are sick, to a preventive strategy, which is individualized.

Big Power, Big Responsibility DNA chips will help scientists make sense of genetic information. Medical applications are on most people’s minds, but the same technologies can be used for everything from confirming lineages of racehorses to teasing out evolutionary relationships between closely related species. But as the power of chips and genetic science grows, questions that society must answer pop up right and left. Should employers use genetic information in hiring decisions? How about insurers who may want to avoid insuring people at high risk for certain diseases? How about a zealous political group trying, say, to portray an opposing candidate as having a high risk of dying of a heart attack? Today, it’s impossible even to list all the questions, let alone answer them. It will take laws, regulations, constraint, and wisdom to ensure that the good consequences of the genetic revolution outweigh the bad, say many researchers. But still, this is one field that is more exciting, or in which it is more important for us all to imagine the future. A hand­held DNA Chip device, made by Nanogen, Inc. The circles at the top are sample ports. The wires guide electric fields over the DNA array, located on the diamond in the centre. A hand­held DNA Chip device, made by Nanogen, Inc. The circles at the top are sample ports. The wires guide electric fields over the DNA array, located on the diamond in the centre.

64 / Class 12 E d u h e a l F o u n d a tio n

A career that is impacted by biotechnology is not just a job. It is an invitation to participate in the development of new products and possesses that could improve the quality of human life as much as any other discovery since the industrial revolution. Scope of Biotechnology Biotechnology as a subject has grown rapidly. And as far as employment is concerned, it has become one of the fast growing sector. Employment record shows that biotechnology has a great scope in future. Biotechnologists can find careers with pharmaceutical companies, chemical, agriculture and allied industries. They can be employed in the areas of planning, production and management of bio­processing industries. There is a large scale employment in research laboratories run by the government as well as the corporate sector. Biotechnology students in India may find work in a government­based entity such as universities, research institutes or at private centers as research scientists/assistants. Alternatively they may find employment in specialized biotechnology companies or biotech­ related companies such as pharmaceutical firms, food manufacturers, aquaculture and agricultural companies. Companies that are engaged in business related to life sciences (ranging from equipment, chemicals, pharmaceuticals, diagnostics, etc.) also consider a biotech degree relevant to their field. The work scope can range from research, sales, marketing, administration, quality control, breeders, technical support etc.;Armed with this powerful combination of fundamental cell and molecular biology and applied science, graduates are well placed to take up careers in plant, animal or microbial biotechnology laboratories or in horticulture, food science, commerce and teaching. Some major organizations employing bio­technologists in India include Hindustan Lever Ltd, Thapar Group, Indo­American Hybrid seeds, Biocon India Ltd, Bivcol, IDPL, India Vaccines Corporation, Hindustan Antibiotics, National Botanical Institute, National Chemical Laboratories, Tata Engineering Research Institute etc. As there is increasing popularity and explosive growth, there is plenty of opportunities available in Biotechnology field. Some of the examples of the jobs in the biotechnology are – Research Scientist, Teacher, Marketing manager, Science Writer, Bioinformists, Quality Control Officer or Production in­charge in the Food, Chemical and Pharmaceutical industry. Analyst (Venture­ Capitalist) Environmental / Safety Specialist. Biotechnology companies require Corporate Executives with business/management Degrees. A graduate in Biotechnology can get job in government sectors such as Universities and Colleges, Research institutes or at Private Centers as Research scientists/assistants – Some of the main research centres where biotechnologists can make career in researches are : 1. Council for Scientific and Industrial Research, New Delhi. 2. National Environmental Engineering Research Institute, Nagpur.

CAREER IN BIOTECHNOLOGY

Class 12 / 65 E d u h e a l F o u n d a tio n

3. Centre for Cellular and Molecular Biology, Hyderabad. 4. National Institute of Immunology, New Delhi 5. Indian Agricultural Research Institute, New Delhi. 6. Department of Biotechnology (DBT). 7. Defence Research and Development Organisation. 8. Department of Science and Technology. 9. National Facility for Tissues and Cell Culture, Pune. 10. Indian Council for Medical Research, New Delhi. 11. Indian Institute of Science, Bangalore, and 12.R & D departments of pharmaceutical companies like Ranbaxy, Zydus cadila, Lupin

Labs, Dr. Reddy’s lab etc. At present there is a shortage of trained people in this field. Therefore the DBT is trying to promote the subject in schools and colleges. Also CBSE introduced it at pre university level and some institutes provide bachelors’ degree in biotechnology. A short­term training for biotechnologists who wish to work in the industry is organised by Biotech Consortium India Ltd.

Eligibility and Courses area If you want to join a course in biotechnology you must have a background in science. • Students having physics or agriculture, chemistry and biology at the intermediate level

can join for Biotechnology. • In India, some universities offer the B.Sc biotechnology which one can join after class

12 or equivalent examination, with physics, chemistry and mathematics/PCB/Sciences. • Graduates in all sciences/engineering technology/medicine are eligible for the

postgraduate (M.Sc.) course in biotechnology. • Programme in Biotechnology. P.G. courses available are M.Sc. Biotechnology, M.Sc.

(Agriculture) Biotechnology, M.V.Sc. (Animal) Biotechnology, M.Tech. Biotechnology, M.Sc./ M.V.Sc.in Veterinary Biotechnology, M.Sc.(Marine) Biotechnology, Medical Biotechnology, M.Tech.in Biomedical Engineering/Biotechnology.

• Depending upon the aptitude and necessity, more advanced courses such Ph.D. and Post­Doctoral Research in Biotechnology can also be pursued.

• Integrated M.Tech. programme offered at IIT Delhi and Kharagpur is through a Joint Entrance Examination (JEE), which is held for students who have successfully completed the 10+2 or equivalent examination, with physics, chemistry and mathematics.

• The Jawaharlal Nehru University, New Delhi, AIIMS, Jadavpur University, Kolkata and Anna University also offers M.Sc. Courses in Biotechnology.

• For admission to IIT, a pass in the IIT­JEE is required. • Selection to the graduate courses ( BE / B.Tech ) is based on merit i.e the marks

secured in the final exams of 10+2 and through entrance exams.

66 / Class 12 E d u h e a l F o u n d a tio n

• Entrance to the IIT's is through JEE (Joint Entrance Exam) and for other institutions through their own separate entrance exams, and other state level and national level exams.

• Apart from the IIT's, some other famous institutes also recognize JEE scores for selection.

• Selection to the postgraduate courses ( M.Sc / M.Tech) in different universities is through an All India Combined Entrance exam conducted by JNU, New Delhi and to IIT's through GATE in Two year/ 4 semester M.Tech courses and through JEE in five year integrated M.Tech courses in Biochemical engineering and Biotechnology.

Duration Three years duration for B.Sc course. Two years to complete the M.Sc. course. B.Tech. course is of four years duration and five year for integrated M.Tech. programme offered at IIT Delhi and Kharagpur. Institutions providing courses in Biotechnology B Tech/BE • IITs at Guwahati/Madras/Roorkee (www.iitr.ac.in)/

Kharagpur (biotechnology and biochemical engg) (www.iitkgp.ernet.in); and IIT Delhi (www.iitd.ac.in) (MTech biochemical engg and biotechnology)

• Delhi College of Engineering (DCE) (www.dceonline.net)

• Manipal academy of Higher Education, Manipal (www.manipal.edu)

• Anna University, Chennai (www.annauniv.edu) • Jaypee University of Information Technology (JUIT),

(HP)(www.jiitindia.org) • Vellore Institute of Technology, Vellore(www.vit.ac.in) Postgraduate • University of Pune, Pune (www.unipune.ernet.in) • Jawaharlal Nehru University, New Delhi (www.jnu.ac.in) if conducts a combined test for

14 universities all over India. • All India Institute of Medical Sciences, New Delhi (www.aiims.ac.in) • Madurai Kamaraj University, Madurai (www.mkuniversity.org) • Pondicherry University, Pondicherry (www.pondiuni.org) • University of Calcutta, Kolkata (www.caluniv.ac.in) • Bharathiar University, Coimbatore (www.bharathiaruni.org) • Department of Management Science, University of Pune (www.dms.unipune.ernet.in)(MBA

Biotech). For more details : attend career workshops conducted by Eduheal Foundation Call EHF Helpline ­ 09350232519, for more details.

BIOTECHNOLOGY CAREER PATH

Phy, Chem & Bio (PCB)

Phy, Chem, Maths (PCM)

Class XII

B.Tech from VIT (entrance)

B.Sc in Biotechnology from various institution/ universities or allied subjects like Biochemistry, Genetics, Microbiology

B.Tech. M.Tech Integrated

B . S c . / B . Te c h (through exams like AIEEE, or Direct Admission)

M.Tech

P.hd Job Ph.D

Apply for M.Sc Biotech

Entrance Exam Like JNU, AIIMS etc.

Marketing

Processing Industry

Teaching

Research Associates

Own Industry

Research &

Development

Class 12 / 67 E d u h e a l F o u n d a tio n

As the pool of genes has diversified through time it has created an enormous body of biodiversity. Biodiversity is eroded away through over­exploitation by humans, including destruction of habitats, as well as through natural changes in the environment. Biotechnology provides several tools that can help preserve biodiversity and diminish the amount of biodiversity lost in these ways. When the last woolly mammoth died out several thousand years ago, the end of the species was definite. It was irreversible because there were no more breeding pairs left. Today biotechnologists are working on bringing back the woolly mammoth. Woolly mammoth carcasses have been found in the ice packs of northern Russia that have very well preserved DNA. Live cells are required for cloning, so right now it is not possible to clone the mammoth, but by using that DNA it may be possible in a decade or so to create another mammoth. This is only possible where the DNA has been preserved.

In the case of the woolly mammoth nature itself preserved the DNA. As species are being depleted today, biotechnology can provide more certain ways of preserving germplasm that otherwise may be lost forever. The first step in preserving biodiversity is documenting what we have and what is most at risk. The following tasks look at various methods that have been applied to save or recreate germplasm that potentially could be lost.

SAFEGUARDING BIODIVERSITY THROUGH BIOTECHNOLOGY

68 / Class 12 E d u h e a l F o u n d a tio n

What is germplasm? “In textbook terms, germplasm is the collective hereditary genetic material of a life form combining the variations of many different, very closely related entities. Essentially, it’s the material that houses an organism’s blueprint for life. Plant germplasm includes genes that determine everything from how a crop yields to how well it stands up to disease. Germplasm has been around for as long as life itself, but the term is distinctly modern, one that has become more common as science has advanced. As plant breeding has taken crop science down to the level of molecules, germplasm has become the term used to describe the raw material that plant breeders manipulate to develop new crop varieties. In crop breeding circles, germplasm used to be thought of mainly as physical plant material ­ something that could be touched and seen. Today, it’s viewed more specifically as the genetics within a plant, independent of the plant itself. This is an indication of the new power of science to identify and work with genes at the molecular level.”

Activity : Special hybrids Sometimes nature has not allowed man to select the best for his needs and develop the organism further on his own. This is the case with the mule. Our ancestors crossed donkeys and horses for various reasons since ancient times but what characterizes them is their stubbornness. This may be because of their high level of intelligence and strong instinct for self­preservation. These traits may make them more difficult to train, but may also make them the dependable, loyal companion that their owners cherish so much. Mules have a diverse history. The Hittites held them as far more valuable than a chariot horse, and the mule was the favoured mount of the kings of Israel in Biblical times. In the Middle Ages, the mule was the chosen mount of the clergy. To keep a stock of mules, horses (Equus caballus) and donkeys (Equus asinus) have been deliberately crossed. A male mule cannot be mated with a female mule to breed more mules the way a good cow and bull can be bred to produce better calves. The horse and the donkey have differing numbers of chromosomes so meiosis in a mule cannot occur properly resulting in infertility. Use the Internet to search for information on mules and then answer the following questions: 1. Find definitions for the following terms; jack, jenny (or jennet), stallion, mare, hinney. 2. By looking at the Latin names for the horse and the donkey, make a statement about the

relationship between the two species. 3. Mules have physical characteristics drawn from both parents but its conformation (i.e. size

and shape) comes from its mother. However some characteristics of mules are always the same. What are some of these characteristics?

4. What are ligers and tigons? After defining them, write a paragraph on each, using information from the Internet.

Class 12 / 69 E d u h e a l F o u n d a tio n

5. Find the Latin names for the parents of ligers and tigons. Make a statement about the relationship between the two species.

6. Are ligers and tigons sterile like mules? 7. What is a li­liger? 8. What are other hybrids of these hybrids called?

For the following two questions you have to think about using the knowledge you have of taxonomy and genetics. You will not find these answers on the Internet.

9. What is the difference in the relationship between the parents of a mule and the parents of a liger?

10.How can the difference in sterility between the hybrids be explained?

Activity : Fast Growing Broilers Man has on occasion changed an organism so much to suit his own desires that it has resulted in cruelty to the animal. A particular case in point will be the way he has tried to get a chicken to grow as big as possible in as short a time as possible to eat. Read the following passage and think about answers to the questions contained in the text. Chickens are bred mainly for two purposes – egg laying capacity and maximum meat mass. Chickens bred for eggs will be selected based on the number of eggs the hen lays in a certain period. Such a hen does not have to be meaty and plump because she serves her purpose through laying eggs. She will earn her keep by laying eggs. Broilers are chickens bred for their meat quality. In order for the farmer to make a profit he has to get a chicken to an edible size as quickly as possible. Broilers will therefore be selected on their speed of growth and stocky shape. The selection for these characteristics has resulted in some broilers growing so quickly that their bones cannot hold their weight and they have severe problems with their legs. Some are so deformed eventually that they cannot walk properly at all. They outgrow their legs, heart and lungs and many suffer and die of heart disease. 1. It can be argued that if the bird is slaughtered at a stage before these complications set in then they are not really a problem. This means that just as the bird reaches adult size, although not yet mature as an adult, it is slaughtered and eaten before it can pick up any problems resulting from being overfed. However, what is the problem the farmer has in terms of his long­ term business if he carries on with this practice? To prevent this from happening, farmers tried to feed the broilers a restricted diet. BUT, one of the effects of breeding for quick growth is at the same time they were bred for their voracious appetites. So now restricted feeding which would be good for the skeletal formation of the broiler actually results in the birds being hungry much of the time. And a hungry bird is an unhappy, stressed bird. 2. When you consider the problem of overfeeding with the problem of trying to keep some breeding stock, what solution would you propose for the broilers welfare? Explain your answer in detail.