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DNA AND GENETIC ENGINEERING

nanonanananonanonanonananananonanana -LECTURE BY PN KAMARIAH HARON-

LH Page 2

Genetic Studies

Nucleic Acids (polynucleotides)

- Biological molecule that is essential for life.

- DNA and RNA genetic materials.

- Nucleotides:

Form building block of DNA and RNA.

Formed by covalent linkage.

Consist of pentose sugar, nitrogenous base and phosphate group.

- The main difference between nucleotide and nucleoside is that it lacks

phosphate group.

RNA

- Normally single stranded

- Genetic material of small viruses (tobacco mosaic virus // TMV)

- Messenger RNA (mRNA)

Carry the genetic information in synthesis of proteins.

- Transfer RNA (tRNA)

Carry amino acid during synthesis of protein.

- Ribosomal RNA (rRNA)

A site of protein translation.

DNA

- Very large macromolecules.

- Two strands of DNA form double helix by hydrogen bonds.

- DNA associated with an array of different proteins to form chromosomes.

- A genome is the complete genetic material of an organisms. (the whole

sequence of DNA and the whole sequence of the bases)

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Double helix DNA

- Double helix structure of DNA was proposed by Watson and Crick (1953)

- Based on discoveries by other scientists.

1. Rosalind Franklin:

An expert in x-ray crystallography.

Used the technique to examine DNA fibers.

DNA has helical structure.

2. Chargaff (1947)

Analyzed DNA composition of different organisms.

Discoveries:

a) DNA composition is specific-specific. Amount of ratios and

nitrogenous bases vary from one species to another.

b) In every species studied, the base ratios was regular.

i.e. number of A=T and number of G=C.

3. Linus Pauling

Ball-and-stick approach.

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DNA model structure (Watson & Crick, 1953)

- Consist of 2 strands of polynucleotide chains coiled together to form a

spiral (double helix)

- Run in opposite direction (anti-parallel) and linked together by H bonds.

- Sugar and phosphate form the backbone outside the helix.

- Nitrogenous base inside the helix.

- Purine bases pair with pyrimidine bases. A-T (2H bonds) and G-C (3H bonds,

stronger that A-T)

- DNA molecule stabilized by:

a) H bonds between paired bases (collectively strong)

b) Van der waals forces between stacked bases.

- DNA helis uniform width = 2nm.

- Adjacent base pairs are 0.34nm apart.

- Helix makes one full turn every 3.4nm (10 nucleotides)

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Base Pairing Rules

- Explain every Chargaff findings.

Ratio of base is regular. Therefore, in a given DNA molecule, amount of

A is equal to amount of T and amount of G is equal to C.

- Maintain consistent separation width between 2 strands of DNA double

helix.

- Provide stability of DNA double helix i.e. H bonds between bases and Van

der Waals forces between stacked bases.

- The sequence of bases is highly variable along the length of a DNA strand.

Suitable for coding genetic information.

- Suggest the general mechanisms for DNA replication.

Since the base pairing are specific, information on one strand

compliments the information along the other strand.

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THE CENTRAL DOGMA CONCEPT

A genetic material (DNA)

- Must be able to store genetic information and transmit it accurately from

generation to generation through replication.

- Must be able to control the phenotype development of an organism (gene

expression) through protein synthesis. (transcription and translation)

DNA(replication)--(transcription)RNA--(translation)Protein

DNA replication

- A process by which a DNA molecule can produce an exact copy of itself.

- Three models for mechanism of DNA replication were proposed:

a) Semi-conservative.

b) Conservative.

c) Dispersive.

Semi-conservative.

- Two parental DNA strands separate.

- Each strands then serves as template for the synthesis of a new DNA strands.

- The result is two DNA double helixes, which consist of one parental strand

and one new strand (half conserved)

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Conservative.

- The two parental DNA strands join back together after replication.

- One daughter molecule contains both parental DNA strands (completely

conserved)

- The other daughter strands contains new DNA strands.

Dispersive

- The parental double helix is broken into double-stranded DNA segments that

act as templates for the synthesis of new double helix molecules.

- The segment of parental and daughter (new) DNA are interspersed in both

strands with segments after replication.

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History

1. Meselson and Stahl.

- Devised experimental approach to distinguish the three mechanisms.

- Proved that replication is semi-conservative.

- Used E. Coli that grown in 15N medium for few generation.

- Then, it is transferred into 14N medium.

- Each generation is separated (after one round of replication)

- It is analysed using density gradient centrifugation.

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DNA replication based on semi-conservative model

- Double helix DNA unwind to form replication fork.

- Catalysed by the enzyme, DNA helicase. (break hydrogen bonds)

- DNA synthesis occurs in 5’-3’ direction using a DNA template strands.

- Two strands of DNA are replicated simultaneously.

a) Leading strand (continuous synthesis 5’-3’ direction towards the

replication fork)

b) Lagging strand (discontinuous synthesis 5’-3’ direction against the

replication fork)

Formation of leading strand

- RNA primer (short length RNA) synthesized by RNA helicase (enzyme) on

template DNA.

- RNA primer allows DNA polymerase to bind nucleosides to the template and

begin replication (5’-3’ direction)

- RNA primase removed.

- DNA polymerase III elongate the strand by adding new nucleoside (from the

nucleoplasm) one by one according to base pairing rules and catalyze the

information of phosphodiester bonds.

Formation of lagging strand

- Synthesis of RNA primer on the antiparallel template strand. (opposite direction of

replication fork)

- DNA polymerase bind to template and begin replication.

- Nucleosides attached to the complementary bases in a 5’-3’ direction to form short

lengths of DNA called Okazaki fragments. Nucleoside joined together catalyzed by DNA

polymerase.

- RNA polymerase are freed from Okazaki fragments by DNA polymerase I.

- Okazaki fragments are joined together by enzyme DNA ligase, forming continuous lagging

strand.

- Replication compete – two double stranded daughter DNA molecules formed. Each has

one parent strand and one new strand.

- The new double-stranded daughter DNA swist to form double helix molecules

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Protein synthesis

- Protein:

Structural and metabolic. e.g. collagen, haemoglobin, etc.

Amino acids arranged in specific sequence.

Determine the physical and chemical nature of protein.

Archibald Garrod (1908)

- First to propose the relationship between genes and production of enzymes

(gene encode enzymes)

- Studied inherited disease alkaptonuria.

- Normal individual produced enzyme homogentisic acid and oxidase that

broke down alkapton (chemical)

- Patient (lack enzyme) alkapton released into urine.

Beadle and tatum (1940)

- Studied bread mold, Neurospora Crassa (exposed to x-ray)

- Mutants are defect in metabolic pathway that synthesize amino acid

arginine.

- Mutants are defective in a single gene (lack a single enzyme)

- Single gene control the synthesize of single enzyme.

One gene-one enzyme hypothesis.

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Gene concept: One gene-one polypeptide hypothesis.

- Modification of hypothesis:

Enzymes are one category of proteins.

All proteins are encoded by genes.

Many proteins are constructed from two or more different polypeptides

and each polypeptide is specified by its own gene.

One gene-one polypeptide hypothesis.

Flow of genetic information

- DNA controls cell activities.

Control various types of proteins.

DNA: nucleotide sequence protein: amino acid sequence

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Molecular gene expression

1. Transcription:

- Produce RNA copy of the gene.

- Information in DNA is transcribed (copied) to mRNA molecule.

- DNA strands act as template.

- mRNA is transcribed complement to the DNA template strand.

- mRNA carry information from DNA to ribosomes.

2. Translation:

- Synthesizing specific polynucleotide on a ribosome.

- Genetic information on the mRNA is translated by ribosomes into amino

acid sequence of polypeptides.

- Requires tRNA to carry a specific amino acid and add it to the growing

polypeptide chain according to the code in mRNA.

Transcription and Translation is called as Central Dogma of Gene

Expression.

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DNA

Protein synthesis

1. Prokaryotes

- No nucleus to separate transcription and translation

- Translation can begin immediately while mRNA is being transcribed

- Both occur in cytoplasm

2. Eukaryotes

- Transcription occurs in nucleus and translation in cytoplasm.

- Pre-mRNA produced, then processed to produce mRNA.

- Mature mRNA exits nucleus into the cytoplasm to be translated.

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Transcription

1. A protein-coding gene codes the synthesis of a specific protein, consist of:

- promoter: a base-pair sequence specifies where transcription begins.

- RNA-coding sequence: a base-pair sequence that include coding

information for polypeptide chain specified by gene.

- terminator: a base pair sequence that specifies the end of the mRNA

transcription.

2. Catalysed by enzyme RNA polymerase – uncoil the protein-coding segment

(a single gene)

3. One strand becomes template (antisense strand). Base sequence of mRNA

is complementary it.

4. The other strand (sense strand has the same sequence of mRNA except

Thymine replaced by Uracil) – protein-coding gene.

5. Activites of protein determine the structure and functions of cells.

6. A protein link between the genotype and the phenotypes of the organisms.

Three stages of transcription

i) Initiation

- At the promoter site.

- Specific recognition by RNA polymerase of promoter base sequence

(prokaryotes) and a complex protein (eukaryotes).

- RNA synthesis initiated – addition of free DNA nucleotides by RNA

polymerase in the 5’-3’ direction.

- Base pairing rule except U replaces T to pair with it.

ii) Elongation

- Addition of more free RNA nucleotides in 5’-3’ direction to form a mRNA

strand i.e. complementary to DNA template (antisense strand).

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iii) Termination

- RNA polymerase recognize the terminator base sequence.

- mRNA strand seperates from RNA and then translated.

*DNA open certain parts during the process and coiled back into double helix

after process. Pre-mRNA is produces during transcription.

RNA base pairing.

mRNA processing

- in eukaryoes, gene contain introns – break up coding sequence, exons.

- Introns is a non-coding sequence, transcribed to pre-mRNA but not

translated.

- Introns are removed and exons are spliced together to produce mature

mRNA.

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Genetic code

- Information of mRNA in code form (genetic code).

- Triplet code: three nucleotides in mRNA specify one amino acids in

protein (codons)

- Nonsense codons: do not code for any amino acids

- AUG-start codon: also codes for amino and methionine.

- UAA, UAG and UGA: terminator codons.

Translation

- Initiated by the assembly of mRNA, tRNA and ribosomal subunit (rRNA)

- Required various types of enzymes such as amino acid – tRNA

synthetase.

- Involved mRNA, tRNA and ribosomal subunit (rRNA)

- Required various types of enzymes such as aminoacyl tRNA synthetase

and ATP.

- Three stages:

a) Initiation.

b) Elongation.

c) Termination.

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Transfer RNA (tRNA)

- Transfer free amino acids from the cytoplasm and arrange them into

polypeptide chain according to the codons on the mRNA.

- Shortest RNA.

- Many parts are complementary to other part – “clover leaf” shape.

- Contain anticodon that complementary to codon on mRNA.

- Amino acid attach to 3’ and of tRNA.

i) Initiation

- mRNA, first aminoacyl tRNA (initiator tRNA) and ribosomal subunits

assemble to form a functional complex.

- Facilitated by ribosomal intiation factors.

- Use energy.

- mRNA bind to small ribosomal subunit.

- Initiator tRNA recognize the start codon AUG mRNA and binds to it.

- Large subunit binds with small subunit.

- Initiator tRNA located in the P site.

*at the 3’ and has methionine.

ii) elongation

- Covalent bonding of amino acids to each other form polypeptide.

a. Charged (aminoacyl) tRNA carrying a single amino acid bind to A site

(use energy).

Anticodon of tRNA are complementary to the codon in the mRNA.

Peptidyl tRNA at the P site.

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b. - Peptide bond formed between amino acid at a site and growing

polypeptide

- polypeptide removed from the tRNA at P site.

- transferred to tRNA at A site.

- the event called peptidyl transferase reaction.

- ribosome moves toward 3’ end (I codon).

- shifted tRNA at P to E and A to P.

- A site empty, ready to accept charged tRNA.

iii) Termination

- When a stop codon (UAA, UAG, and UGA) is found at the A site.

- Recognized by release factors that mimics the structure of tRNA.

- Release factor binds to stop codon.

- Bond between polypeptide and tRNA hydrolysed.

- Both released to form ribosome.

- Ribosomal subunits, mRNA and release factor dissociate.

Polyribosome. (Polysome)

- Translation can involve more than one ribosome.

- mRNA attach a group of ribosome and translation occur

simultaneously.

- Each ribosome will produce one polypeptide that is similar.

- Produce a number a polypeptides at a time.

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Genetic Engineering

Introduction:

- Also called genetic modification.

- The alteration genetic code by artificial mean.

- Used various technique and procedures in gene manipulation.

- Modify DNA and recombine DNA (protein/animal/plant with desirable

traits)

- Modify DNA and recombine DNA ( protein/animal/plant with desirable

traits)

- Application in medicine, forensic, agriculture, and pharmaceuticals.

Recombinant DNA technology

1. DNA recombination : DNA segment moves from one DNA molecule to

another randomly.

- During meiosis 1 through crossing over of homologous chromosomes.

- During fertilization when two gametes fuse, or

- Through mutation.

2. By using DNA recombinant technology:

- DNA recombination can be directed to serve specific purpose for

instance the mass production of human insulin by bacteria.

- In order for bacteria to produce human insulin, the bacterial gene must

be modified by inserting the human insulin gene into it.

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Basic procedures of recombinant DNA technology

Stage I: isolation of the required/target gene.

|

Stage II: insertion of the gene into a vector.

|

Stage III: introduction of gene into host using vector.

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Stage IV: cloning of gene by the host cells.

Material used in recombinant DNA technology

- The target gene.

- Cloning vectors: plasmid and bacteriophage to carry target gene into

host cell.)

- Restriction enzyme used to cut DNA into fragments.

- Modifying enzyme (DNA ligase to join DNA fragments together)

- Host cell (bacterial cell that allows cloning bector to replicate within it)

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Stage I: Isolation of target gene

- Using two methods:

a) Cutting the gene from a complete chromosome.

b) Producing a complementary DNA (cDNA)

a) Cutting the gene from a complete chromosome

- There are four basic steps in a DNA extraction:

The cell distruption or cell lysis (exposing DNA)

Removing membrane lipids (using detergent)

Removing protein by adding a protease or phenol – chloroform

extraction.

Precipitating the DNA with an alcohol (ice-cold) producing pellets

upon centrifugation

DNA can be resolubilised in a slightly alkaline buffer or in ultra-

pure water.

b) Producing Complementary DNA (cDNA)

- Produced through reverse transcription.

- mRNA acts as template to synthesize DNA using reverse transcriptase

enzyme.

- The mRNA of the target gene is extracted from the cells where the gene

is actively synthesizing protein.

- cDNA is an intron-less DNA. (Example: cDNA for insulin gene can be

extracted from the pancreas)

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Restriction enzyme digestion

- digest target DNA and vector.

- Produce fragments of various lengths (restiriction enzyme)

Restriction enzyme

- Naturally found in bacteria that are resistant to bacteriophage infection.

Act as defense mechanism.

- Cut or cleave bacteriophage DNA when they are inserted into cell.

- As a result, the phage DNA is not functional (restricted)

- Bacteria protect its DNA from being digested by adding methyl group

(-CH3) to codeine or cytosine within the restriction site – methylation.

- Very specific - recognize a specific base sequence (restriction site) and

cut as a specific point between two nucleotides.

- Restriction sequence consist the same four to eight nucleotides on both

DNA strands but arranged in opposite direction – palindromic

- Very useful tool in genetic engineering.

- Most restriction enzyme cut DNA in staggered way, results in DNA

fragments with unpaired bases on the end – sticky ends.

- Unpaired bases form hydrogen bonds with complementary bases of the

sticky end at other DNA fragments/target gene.

- Join two DNA of diggerent sources to produce a recombinant DNA.

- Other type of restriction enzymes cut DNA in a straight way – blunt end

fragments.

- Different restriction enzyme recognize different restriction site.

- Produce a range of different size of fragments (restriction fragments)

- Easier to study/insert a smaller or shorter DNA fragments

Examples of restriction enzymes include:[46]

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Enzyme Source Recognition Sequence Cut

EcoRI Escherichia coli

5'GAATTC

3'CTTAAG

5'---G AATTC---3'

3'---CTTAA G---5'

EcoRII Escherichia coli

5'CCWGG

3'GGWCC

5'--- CCWGG---3'

3'---GGWCC ---5'

BamHI Bacillus amyloliquefaciens

5'GGATCC

3'CCTAGG

5'---G GATCC---3'

3'---CCTAG G---5'

HindIII Haemophilus influenzae

5'AAGCTT

3'TTCGAA

5'---A AGCTT---3'

3'---TTCGA A---5'

TaqI Thermus aquaticus

5'TCGA

3'AGCT

5'---T CGA---3'

3'---AGC T---5'

NotI Nocardia otitidis

5'GCGGCCGC

3'CGCCGGCG

5'---GC GGCCGC---3'

3'---CGCCGG CG---5'

HinfI Haemophilus influenzae

5'GANTCA

3'CTNAGT

5'---G ANTC---3'

3'---CTNA G---5'

Sau3A Staphylococcus aureus

5'GATC

3'CTAG

5'--- GATC---3'

3'---CTAG ---5'

PovII* Proteus vulgaris

5'CAGCTG

3'GTCGAC

5'---CAG CTG---3'

3'---GTC GAC---5'

SmaI* Serratia marcescens

5'CCCGGG

3'GGGCCC

5'---CCC GGG---3'

3'---GGG CCC---5'

LH Page 24

Stage II: insertion of gene into a vector.

- The target gene is then inserted into vectors (gene carrier) such as

bacterial plasmids or bacteriophages DNA to produce recombinant DNA,

combining DNA from two sources.

- Vector is a DNA molecule used as a vehicle to transfer foreign genetic

material into another cell.

- Vector must process marker genes such as amp R (resistant to

amphicillin)

- Able to carry foreign DNA into a host cell.

- Most commonly used cloning vectors are bacterial plasmids, other

vectors are viral phages, cosmids and yeast artificial chromosomes

(YAC).

Bacterial Plasmids (pUC 18)

- Small, double stranded circular DNA found in most bacteria.

- Able to replicate independently from the bacterial chromosome.

- Contain a replicator or “ori” (the origin of replication)

- Must contain genetic markers for selection such as amp R (resistant to

amphicillin)

- Must be able to replicate freely and rapidly.

- Must be able to accept foreign DNA in multiple cloning site (MCS) i.e. they

possess many restriction sites for many restriction enzymes.

- Plasmid + foreign gene is known as recombinant plasmid are inserted into the

host cell by transformation.

- Able to carry 10-15 thousand base pair (10-15kb)

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Bacteriophages

- viruses infected bacteria (lambda 2001)

- linear double helix DNA.

- A part of the bacteriophage DNA which is not required for replication is

removed and replaced with the target gene.

- The recombinant DNA (rDNA) produce.

- Returned bak into a bacteriophage and inserted into a host cell i.e.

bacteria through infection.

- The process is called “Transfection”

- rDNA replicates in bacteria (E. coli)

- Able to carry up to 25kb of foreign/target DNA.

- High transformation efficiency

- About 1000 times more efficient than the plasmid vector.

*notes: Nucleoside = sugar + base

Nucleotide = sugar + base + phosphate

Cosmids

- Cosmids are plasmid vectors that contain cos sites. The cos site is the only

requirement for DNA to be packaged into a phage particle

- Example: s COS-1

- A combination/hybrid of bacteriophage and plasmid.

- Infect host cell in a phage-like manner and transfer rDNA into host.

- Able to carry up to 45kb target DNA.

- High transformation efficiency

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Figure: cosmid Figure: Yeast artificial Chromosome (YAC)

Yeast Artificial Chromosome (YAC)

- Eukaryotic chromosome (yeast) and foreign DNA

- Capable of carrying a large DNA fragment (up to 2Mb)

- But its transformation efficiency is very low.

Modifying enzymes

- DNA ligase - joins the target gene and the vector to product recombinant

DNA.

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DNA recombinant (rDNA)

- The vector must be digested with the same restriction enzyme.

- The exposed bases on sticky end will attract complementary bases of other

sticky end and from hyrdrogen bonds.

- DNA fragments are then connected/joined by DNA ligase.

- Form covalent bond between the nucleotide producing recombinant DNA.

- Produce two types of vectors,

Vectors without target gene (reannealing of sticky ends)

Recombinant DNA with target gene.

Reannealing is the process by which two single strands of DNA combine to

form double-stranded DNA.

Stage III: Introduction of the gene into a host using vectors.

- The recombinant DNA is introduced into a host cell to replicate.

- A host can be prokaryotic cell e.g. bacteria or eukaryotic cell

(yeast/animal cell)

- Carried through transformation by plasmid or transfection (infection) by

phage DNA.

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Characteristic of the host cell

- Must be able to accept foreign DNA i.e. recombinant DNA.

- Must be able to maintain the same structure of the recombinant DNA

from one generation to another.

- Must be able to amplify the gene of the recombinant DNA.

- The recombinant plasmids must be introduced into a host cell (e.g. E.

coli) through transformation.

- The mixture of two types of plasmids are mixed with E.coli in a medium

containing calcium chloride.

- Ca2+ induced the permeability of the cell wall, make it able to take up the

plasmid from the surrounding medium.

- Small number of bacteria take up the plasmids, only a small fraction of

these bacteria contain the recombinant plasmid.

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Stage IV: Cloning of gene by the host cell

- The host cell (e.g. E coli) cultured in a restriction medium (e.g.

containing amphicillin and the sugar X-gal) to grow.

- The transformed E.coli which take up the plasmids will be able to grow

and form colonies on the medium.

- Each time the bacteria divide , the inserted gene will also replicate.

- The bacteria containing the plasmid with infect lac Z gene are able to

produce the enzyme β-galactosidase which can hydrolyse X-gal.

- This will produce a blue compound which makes the colony blue.

- The bacteria which contain recombinant plasmid will form white

colonies because the lac Z gene is not functional.

- The white colonies are then cultured in large scale.

- Each time the bacterial cells divide, the recombinant plasmid will also

replicates, producing multiple copies of bacteria i.e. bacterial clones,

each carrying a copy of the required gene (gene clone)

- The target genes will be expressed to produce gene products (e.g

insulin)

- This stage is referred as gene cloning.

Gene Library

- Collection of cloned DNA fragments.

- Two types: genomic library and cDNA library.

Genomic Library

- Genomic: refers to a complete sets of genes of a particular organisms.

- Genomic library is a collection of a bacterial or viral clone that carry the recombinant

DNA.

- Each clone a particular fragment from a species genome.

cDNA library

- A collection of bacterial or viral clone that carry recombinant DNA with cDNA fragments.

- cDNA library represents only a part of species genome.

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Application of Gene Technology

- Produce transgenic organism / genetically modified organism –

agriculture. (to improve the productivity of plants and animals of

agricultural importance)

- For DNA fingerprinting in forensic investigations.

- Medicine/pharmaceuticals.

Transgenic Organisms

- Organism that are genetically modified or altered i.e. contain foreign

DNA using genetic engineering methods.

- For example:

Bacteria E. Coli made to produce synthetic human insulin.

Cheese production: yeast are made to produce chymosin to

replace the natural enzyme rennin (rennit) that is used to

coagulate milk.

Pseudomonas bacteria are made to break hydrocarbons in oil

which is used to clean up oil spills.

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Transgenic plant

- Genetically modified by genetic engineering technique.

- Produce specific desirable traits.

- DNA technology is widely used in plants.

- No real distinction between somatic cells and germinal cell.

- Root cells grown in culture can be transformed into laboratory with the

desired genes.

- Grown into mature plants with flowers.

- Recombinant DNA is introduced into the plant by :

ballistic method (particle gun) or

by agrobacterium tumefaciens mediated transformation.

- rDNA will combine with plant genome.

Balistic method

- DNA bound to tiny particles of gold or tungsten.

- “shot” into plant tissue or single cell under high pressure.

- Penetrate both the cell wall and membranes.

- DNA separates from metal.

- Integrated into the plant genome inside the nucleus.

- Applied successfully in monocots like wheat and maize.

- Major disadvantage : expensive, serious fdamage to cellular tissue.

Agrobacterium Tumefaciens mediated transformation.

- Causes crown gall disease in plant. (formation of tumour)

- Able to transfer genes by tumour – inducing plasmid.

- Best cloning vector for plant.

- Ti plasmid combined with target gene

- The disease-causing ability is eliminated.

- Ablity to transfer genetic material is not damaged.

- Infection by Agrobacterium Tumefaciens only limited to dicotyledons

(e.g. potatoes)

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Achievements:

1. Production of rice that enabled to manufacture beta-carotene.

2. Toxin gene can be introduced into the plant genome, expressed and

provide resistance against plant viruses.

3. Genes that provide resistance against plant viruses have been

successfully introduced into tobacco, tomatoes and potatoes.

4. Biopharmaceuticals:

- Human growth hormone with the gene inserted into chloroplast DNA

of tobacco plant.

Transgenic animals

- A transgenic animal carries a foreign gene inserted into its genome.

- Examples:

1. Transgenic sheep (1985) and goats express foreign proteins in their

milk.

2. Sheep and goat are used to produce pharmaceutical proteins in

milk.

e.g. AAT protein (mutation of AAT enzyme gene (α-1 antitrypsin)

cause emphysema in humans)

3. Transgenic chicken synthesizes human protein in the “white” of the

eggs.

4. Transgenic mice (1981) become tools for exploring many biological

questions.

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Creating a transgenic animal

Creating a transgenic plant

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DNA Fingerprinting

- Used in parent testing and forensic investigation.

- Assist in the identification of individuals by comparing their DNA

profiles.

- Detect highly variable repeat sequence, variable number tandem

repeats (VNTR)

- Pattern of bonds are visible by using gel electrophoresis.

- Different person has different DNA base sequence except identical twins

(twins have unique pattern of bonds = DNA fingerprinting)

- About 95% of the human DNA consist of non-coding region (introns)

- 30%-40% of introns consist of short sequence of bases which are

repeated many times (VNTR). Some are scattered and some are found

together in clusters.

- Large amount of DNA is needed (PCR is used to amplify DNA.)

DNA profiling process.

- A sample of an individual’s DNA is collected by the use of a buccal swab.

- Create the individual’s DNA profile by using:

Restriction fragment length polymorphism (RFLP) analysis.

a) Earliest technique.

b) Involved RE digestion, followed by southern blot analysis.

c) Analayze VNTR loci.

d) Laborious, and requires large amount of undegraded sample of

DNA.

Figure: Buccal Swab

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Polymerase chain reaction (PCR analysis)

- A method to produce many copies (replication) of specific/target DNA

fragments in vitro.

- Required a thermocycle, microcentrifuge tubes, template DNA, Taq DNA

polymerase ( thermostable), primers and nucleotides.

- Very small/degraded starting samples.

- Analyzing VNTR loci. The US FBI has standardized a set of 13 VNTR way

for DNA typing.

- Denaturation:

Heating the reaction to 94 °C-98 °C for 20-30 seconds. Hydrogen

bonds are broken producing single-stranded DNA molecules.

(separation)

- Annealing:

Temperature reduced to 50 °C-65 °C for 20-40 seconds. Primers

anneal to single-stranded DNA template. Polymerase binds to the

primer-template hybrid and begin DNA synthesis.

- Extension/Elongation:

The temperature at this step depends on the DNA polymerase

used (e.g. Taq polymerase : 72 °C)

- At this step, the DNA polymerase synthesizes a new DNA strand

complementary to the DNA template strand by adding deoxynucleotide

triphosphate (dNTPs) that are complementary to the template in 5’-3’

direction.

- At its optimum temperature, the DNA polymerase will polymerize a

thousand bases per minute.

- Final elongation:

This single step is occasionally performed at a temperature of

70 °C-74 °C for 5-15 minutes after the last PCR cycle to ensure

that any remaining single-stranded DNA is fully extended.

- Final hold:

This step at 4 °C-15 °C for an indefinite time may be employed for

short-term storage of the reaction.

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PCR

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Short tandem repeat (STR) of VNTR analysis

- Uses highly polymorphic region that have short repeated sequence of

DNA (3,4 or 5 bases)

- Many STR regions will be tested at the same time.

- The STRs in use today for forensic analysis are all tetra-nucleotide or

penta-nucleotide repeats (4 or 5 repeated nucleotides) as these give a

high degree of error of data.

Gel Electrophoresis

- Separate the digested/amplified fragments according to their sizes.

- Separation based on the rate of movement through a gel under the

influence of an electric field.

- Move from –ve to +ve pole.

DNA Probes

- A fragment of DNA or RNA of variable length (usually 100-1000 bases

long)

- Complementary to target gene.

- Radioactive-labelled.

- Single stranded.

- May be synthesized in the laboratory.

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Uses of gene therapy in medicine

- Diagnosis (genetic screening) gene therapy.

- DNA analysis carried out using DNA extracted from cells.

- Cells obtained from fetus by chorionic villus sampling or amniocentesis

(amnion fluid test), from child or adult using blood sample or

mouthwash.

- Identify disease-causing gene and heterozygote carriers to prevent

disease.

- Gene therapy is undergone to replace faulty gene with normal gene.

- There are two types of gene therapy:

1. Germ-line therapy

Germ cells are modified

Functional genes are introduced.

It is then integrated into their genomes.

Gene would be passed on the next generation.

Highly effective in counteracting genetic disorder and

hereditary disease.

Proposal rejected due to ethical purpose.

2. Somatic cell therapy

Done by changing some of the somatic cells (non-sex cells)

Changes cannot be inherited.

Only treated people will be cured but the faulty gene will still

be passed to the next generation.

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Example of gene therapy

a. Cystic fibrosis (CF)

- Caused by autosomal recessive allele for cystic fibrosis transmembrane

conductance regulator (CFTR) gene.

- Affected person produces mucus that is too thick and unable to flow

properly.

- Respiratory pathways and lungs become partially blocked, causing

difficulty in breathing.

- People with CF rarely live beyond the age of 30.

Therapy is done by:

Cloning the healthy CFTR genes. Two types of vectors are used to

introduce genes into cells of patients:

i) Genes inserted into harmless virus.

ii) Genes are wrapped in lipid and sprayed down into the lungs

of patient. Lipid packages (liposomes) are absorbed into the

epithelial cells lining of the lung

iii) New gene are expressed allowing epithelial cell to produce

normal mucus.

b. Severe Combined Immunodeficiency Disease (SCID)

- Disease affect gene coding for the enzyme ADA (adenosine deamine)

- Mutant gene is recessive and cannot produce enzyme ADA ( enzyme

ADA is needed by the white blood cell which is responsible for immunity

to infection.

- Patient can live up to 2 years

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Therapy is done by:

Cloning the healthy ADA genes and then insert in into harmless

virus.

The virus will inject the ADA gene into stem cells taken from bone

marrow of the affected baby.

The stem cell which has been modified are cultured and will be

injected back into baby’s bone marrow.

Ethical and Social Issues.

1. Safety

- Hazardous new pathogens escape from lab.

- New medical product – what is the potential harmful side effects?

- Genetically modified food may contain new proteins that are toxic or

cause severe allergies

2. Intelectual property

- Mapping of human genome contribute to advance in gene therapy but:

Who should have the right to examine someone else’s gene?

How do we weigh the benefits of gene therapy against assurance

that the gene vectors are safe.

3. Impact on environment

- Genetically engineered organisms used to clean oil spillage but what is

their potential impact on native species?

- GM plants may become superweeds resistant to herbicides, disease and

insect pest.

4. Ethics

- Germ-cell gene therapy and cloning in production of animals e.g. Dolly

and human cloning.