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III YEAR – V SEMESTER
COURSE CODE: 16SMBEZO1:1
BIOTECHNOLOGY
Dr. R. JENNI
ASSISTANT PROFESSOR OF ZOOLOGY
UNIT – I
I-1- DEFINITIONS AND LANDMARKS IN THE HISTORY OF BIOTECHNOLOGY
The technology uses living organisms to produce and improve
desired products or to manipulate the environment and that
manipulates plants, animals and microbes is popularly known as
Biotechnology.
It deals with the integral applications of Microbiology, Biochemistry,
Plant sciences, and process engineering techniques in manufacturing
and service industries.
The biotechnological processes usually operate at low temperatures,
consume least amount of energy and petroleum fuels and do not
exhaust much pollutants in the environment.
Biotechnology was first recognized as a separate branch by Leeds
City Council in United Kingdom in 1920.
However, the name biotechnology has come into wide use after
1970’s.
The European Federation of Biotechnology (EFB) 1978 defined
biotechnology as “ the integral application of knowledge and
techniques of chemistry, microbiology, genetics and chemical
engineering to draw benefits at the technological level from the
properties and capacities o microorganisms and cell cultures”.
The Organization for Economic Co-operation and Development
(OECD) 1891 defined as the application of scientific and engineering
principles to the processing of materials by biological agents to
provide goods and service”.
The International Union of Pure and Applied Chemistry (UPAC)
defined it as, “the application of biochemistry, biology, microbiology
and chemistry, biology, microbiology and chemical engineering to
industrial process and product and on environment.
M.D. Trevan (1988) defined as “the application of biological
organisms, systems or processes to manufacturing and service
industries.
BIOTECHNOLOGY AND ITS BRANCHES
Biotechnology has adopted techniques of biological sciences,
chemistry, physics, mathematics and computer science. It is divided
into several disciplines.
Tissue Culture Technology : it deals with the culture of cells or tissues
of plants and animals in chemically defined media.
Pharmaceutical Technology : it is concerned with the production of
monoclonal antibodies, interferons, vaccines, toxoids, human growth
hormones, etc.
Recombinant DNA Technology : it deals with the insertion of desired
genes into host cells for manipulating the host DNA.
Agricultural biotechnology: it includes all technologies of crop
improvement, and the application of biofertilizers and selective
biocides in agriculture.
Food biotechnology : it is concerned with preparation, preservation
and utilization of various food items
Fermentation technology : it deals with the culture of cells or
microbes in fermenters to produce alcohols, biogas, organic acids
enzymes, antibiotics, etc.
Mining and metal biotechnology: it is concerned with the use of
microbes in mining and extraction of metals from ores
Environmental biotechnology : it deals wit waste recycling, compost
making and microbial treatment of pollutants which ae otherwise
non-biodegradable.
Industrial biotechnology: it deal with the industrial production of
desired goods. Really speaking it is the applied part of fermentation
technology.
SCOPE OF BIOTECHNOLOGY
Biotechnology basically aims at improving the quality of human life
and at protecting him from dangerous disease
To produce more food for the growing population using the available
land
To raise disease resistant high yielding varieties of crops
To introduce harmless biofertilizers instead of chemical fertilizers
To introduce biocides in agriculture
To preserve germplasm of plants, animals and microbes
To produce pharmaceutical products to treat severe diseases in man
and animals
To produce biofuels for reducing the felling of forest trees for fuel
wood
To make use of various microorganisms in food making and
preservation of the food.
MAJOR AREAS OF BIOTECHNOLOGY
Biotechnology has adopted techniques of biological sciences,
chemistry, physics, mathematics and computer science. It is divided
into several disciplines.
Tissue Culture Technology : it deals with the culture of cells or tissues
of plants and animals in chemically defined media.
Pharmaceutical Technology : it is concerned with the production of
monoclonal antibodies, interferons, vaccines, toxoids, human growth
hormones, etc.
Recombinant DNA Technology : it deals with the insertion of desired
genes into host cells for manipulating the host DNA.
Agricultural biotechnology : it includes all technologies of crop
improvement, and the application of biofertilizers and selective
biocides in agriculture.
Food biotechnology : it is concerned with preparation, preservation
and utilization of various food items
Fermentation technology : it deals with the culture of cells or
microbes in fermenters to produce alcohols, biogas, organic acids
enzymes, antibiotics, etc.
Mining and metal biotechnology: it is concerned with the use of
microbes in mining and extraction of metals from ores
Environmental biotechnology : it deals wit waste recycling, compost
making and microbial treatment of pollutants which ae otherwise
non-biodegradable.
Industrial biotechnology: it deal with the industrial production of
desired goods. Really speaking it is the applied part of fermentation
technology.
SCOPE OF BIOTECHNOLOGY
Biotechnology basically aims at improving the quality of human life
and at protecting him from dangerous disease
To produce more food for the growing population using the available
land
To raise disease resistant high yielding varieties of crops
To introduce harmless biofertilizers instead of chemical fertilizers
To introduce biocides in agriculture
To preserve germplasm of plants, animals and microbes
To produce pharmaceutical products to treat severe diseases in man
and animals
To produce biofuels for reducing the felling of forest trees for fuel
wood
To make use of various microorganisms in food making and
preservation of the food.
GENE TRANSFER TECHNIQUES
Definition
Bacteria reproduce by the process of binary fission. In this process, the chromosome
in the mother cell is replicated and a copy is allocated to each of the daughter cells.
As a result, the two daughter cells are genetically identical. If the daughter cells are
always identical to the mother.
Recombination
Genetic recombination refers to the exchange between two DNA molecules.
Genetic Transfer
Genetic transfer is the mechanism by which DNA is transferred from a
donar to a recipient.
Once donar DNA is inside the recipient, crossing over can occur.
The result is a recombinant cell that has a genome different from either the
donar or the recipient.
In bacteria genetic transfer can happen three ways:
1. Transformation
2. Transduction
3. Conjugation
Transformation
After death or cell lyses, some bacteria release their DNA into the
environment.
Other bacteria, generally of the same species, can come into contact with
these fragments, take them up and incorporate them into their DNA by
recombination.
This method of transfer is the process of transformation.
Any DNA that is not integrated into he chromosome will be degraded.
The genetically transformed cell is called a recombinant cell because it has a
different genetic makeup than the donar and the recipient.
All of the descendants of the recombinant cell will be identical to it.
In this way, recombination can give rise to genetic diversity in the population.
Griffith's Experiment
The transformation process was first demonstrated in 1928 by Frederick
Griffith.
Griffith experimented on Streptococcus pneumoniae, a bacteria that causes
pneumonia in mammals.
When he examined colonies of the bacteria on petri plates, he could tell that
there were two different strains.
The colonies of one strain appeared smooth.
Later analysis revealed that this strain has a polysaccharide capsule and is
virulent, that it, it causes pneumonia.
The colonies of the other strain appeared rough.
This strain has no capsules and is avirulent.
When Griffith injected living encapsulated cells into a mouse, the mouse died
of pneumonia and the colonies of encapsulated cells were isolated from the
blood of the mouse.
When living nonencapsulated cells were injected into a mouse, the mouse
remained healthy and the colonies of nonencapsulated cells were isolated
from the blood of the mouse.
Griffith then heat killed the encapsulated cells and injected them into a
mouse.
The mouse remained healthy and no colonies were isolated.
The encapsulated cells lost the ability to cause the disease.
However, a combination of heat-killed encapsulated cells and living
nonencapsulated cells did cause pneumonia and colonies of living
encapsulated cells were isolated from the mouse.
How can a combination of these two strains cause pneumonia when either
strand alone does not cause the disease?
If you guessed the process of transformation you are right!
The living nonencapsulated cells came into contact with DNA fragments of
the dead capsulated cells.
The genes that code for thr capsule entered some of the living cells and a
crossing over event occurred.
The recombinant cell now has the ability to form a capsule and cause
pneumonia.
All of the recombinant's offspring have the same ability.
That is why the mouse developed pneumonia and died.
Transduction
Another method of genetic transfer and recombination is transduction.
This method involves the transfer of DNA from one bacterium to another
with the use of a bacteriophage (phage).
A phage is a virus that infects bacteria.
The phage T4 and the phage lambda, for example, both infect E. coli.
Because the phage reproductive system is important to understanding
transduction, we will briefly review phage lifecycle.
Phages are obligatoryintracellular parasites and must invade a host cell in
order to reproduce.
T4 multiplies by the lytic cycle which kills the host and lamba multiplies by
the lysogenic cycle which does not cause the death of the host cell.
In lysogeny, the phage DNA remains latent in the host until it breaks out in a
lytic cycle.
General Steps Of The Lytic Cycle:
1. Attachment of T4 to receptors on E. coli cell wall.
2. Penetration of the cell wall by tail core. Inject DNA into host.
3. E. coli DNA is hydrolyzed. Phage DNA directs biosynthesis of viral
parts using the host cell's machinery.
4. The phages mature as the parts are assembled.
5. Lyses of E. coli and release of the new phages.
General Steps Of The Lysogenic Cycle:
6. Phage attaches to E. coli and injects DNA.
7. Phage circularizes and can enter either the lytic or the lysogenic cycle.
8. The lytic cycle would occur as previously described.
9. In the lysogenic cycle the circular phage DNA recombines with E.
coli DNA and the phage DNA is now called prophage.
10. E. coli undergoes cell division, copying prophage and passing to
daughter.
With more divisions there are more cells with the prophage.
11. The prophage may exit the chromosome and start a lytic cycle at any
time.
Now that you have reviewed phage lifecycles, we can discuss transduction.
Transduction can be generalized or specialized.
The Steps Of General Transduction:
1. A phage attaches to cell wall of bacterium and injects DNA.
2. The bacterial chromosome is broken down and biosynthesis of phage
DNA and protein occurs.
3. Sometimes bacterial DNA can be packaged into the virus instead of
phage DNA.
4. This phage is defective (can't destroy another host cell) because it does
not carry its own genetic material.
5. The cell lyses, releasing viruses.
6. The phage carrying bacterial DNA infects another cell.
7. Crossing over between donor and recipient DNA can occur producing
a recombinat cell.
8. In generalized transduction, any bacterial genes can be transferred
bacause the host's chromosome is broken down into fragments.
9. Whatever piece of bacterial DNA happens to get packaged within the
phage is the genetic material that will be transferred between cells.
10. In specialized transduction, on the other hand, only certain bacterial
genes can be transferred.
11. These genes, as you will see, must exist on either side of the prophage.
12. Specialized transduction requires a phage that uses the lysogenic cycle
for reproduction.
The Steps In Specialized Transduction:
1. Remember that in the lysogenic cycle, phage DNA cn exist as a
prophage integrated in the bacterial chromosome)
2. Occasionally when the prophage exits it can take adjacent bacterial
genes with it.
3. The phage DNA directs synthesis of new phages.
4. The phage particles carry phage DNA and bacterial DNA.
5. The cell lyses, releasing the phages.
6. A phage carrying bacterial DNA infects another cell.
7. The joined phage and bacterial DNA circularize.
8. Along with the prophage, bacterial DNA integrayes with the recipient
chromosome by a cross over event.
9. This forms a recombinant cell.
Conjugation
A third mechanism by which genetic transfer takes place is conjugation.
This mechanism requires the presence of a special plasmid called the F
plasmid.
Therefore, we will briefly review plamid structure before continuing.
Plasmids are small, circular pieces of DNA that are separate and replicate
indepentently from the bacterial chromosome.
Plasmids contain only a few genes that are usually not needed for growth and
reproduction of the cell.
However, in stressful situations, plasmids can be crucial for survial.
The F plasmid, for example, facilites conjugation.
This can give a bacterium new genes that may help it survive in a changing
environment.
Some plasmids can integrate reversibly into the bacterial chromosome.
An integrated plasmid is called an episome.
Bacteria that have a F plasmid are referred to as as F+ or male.
Those that do not have an F plasmid are F- of female.
The F plasmid consists of 25 genes that mostly code for production of sex
pilli.
A conjugation event occurs when the male cell extends his sex pili and one
attaches to the female.
This attached pilus is a temporary cytoplasmic bridge through which a
replicating F plasmid is transferred from the male to the female.
When transfer is complete, the result is two male cells.
The F plasmid can behave as an episome.
When the F+ plasmid is integrated within the bacterial chromosome, the cell
is called an Hfr cell (high frequency of recombination cell).
The F plasmid always insetrs at the same spot for a bacterial species.
The Hfr cell still behaves as a F+ cell, transferring F genes to a F-cell, but
now it can take some of the bacterial chromosome with it.
Replication of the Hfr chromosome begins at a fixed point within the F
episome and the chromosome is transferred to the female as it replicates.
Movement of the bacteria usually disrupts conjugation before the entire
chromosome, including the tail of the F episome can be transferred.
Therefore, the recipient remains F- because the F plasmid is not entirely
transferred.
A cross over event can occur between homologous genes on the Hfr fragment
and the F- DNA.
Pieces of DNA not recombined will be degraded or lost in cell division.
Now the recombinant genome can be passed on to future generations.
UNIT – II
RESTRICTION ENDONUCLEASES
The nuclease enzyme that cuts the DNA at a unique sequence, is called
restriction endonucleases
They cut the DNA in a non-terminal(end) region. Restriction
endonucleases are used to generate rejoinable DNA fragments
They are also known as molecular knives, molecular scissors, restriction
enzymes or molecular scalpels.
The sequence recognized by the restriction enzyme to cut the DNA is
called restriction site, restriction endonucleases site or recognition site.
The recognition site consists of 4-8 base pairs.
The enzyme breaks two phosphodiester bonds, one in either stand of the
duplex DNA to cut the DNA. The 3’ cut end has a free OH group and the 5’
cut end has phosphate group.
Some restriction enzymes recognize palindromic sequence to cut DNAs,
but some other recognize non palindromic sequences.
The genome of an organism has several restriction sites for one restriction
enzyme.
The distance between two adjacent restriction sites varies greatly.
So a restriction enzyme produces several DNA fragments of different
lengths while cutting the DNA.
TYPES OF RESTRICTION ENZYMES
The restriction endonucleases are grouped into three types. Thry are
Type I restriction endonucleases
Type II restriction endonucleases
Type III restriction endonucleases
The type I and type III restriction enzymes recognize specific sequence in
the duplex DNA but cut the DNA far away from the recognition sites.
So they are not useful for genetic engineering.
The type II restriction endonucleases recognize specific sites and cut the
DNA at the recognized sites.
So they are of much use in genetic engineering. Eg. EcoRI. Hind III, etc.
S.No. Type I Restriction endonucleases Type II restriction endonucleases
1 The enzyme is made up of three non-identical sub-units
The enzyme is made up of two identical sub-units
2 Molecular weight is 400000 fslyond Molecular weight ranges from 20000 to 00000 daltons.
3 The enzyme has both endonucleases activity and methylase activity.
Restriction activity alone.
4 The site of cutting is 1000 nucleotides away from the recognition site.
The ite of cutting is the same recognition site.
5 The sequence of cutting is non-specific.
The sequence of cutting is specific
6 The enzymes protect DNAs by methylation.
No methylation activity.
7 ATP Mg2 adenosyl methionine are for activation.
Mg2 alone is required for activation.
TYPE III RESTRICTION ENDONUCLEASES:
A type II restriction endonucleases recognizes a specific sequence in the
duplex DNA and cuts the DNA at the recognized sequence.
So the cutting is sequence specific.
The enzyme consists of two identical sub-units and its molecular weight
ranges from 20000 to 100000 daltons.
It requires Mg2 as co-factor for the enzyme activity.
Eco RI, Hind III, Mbo I, etc. are examples for type II restriction enzymes.
At present, about 350 type II restriction endonucleases are isolated from
various bacterial strains.
They are named using the first letter of the genus name, the first two
letters of the species name and the first letter of the strain from which
the enzyme was isolated.
If there are nore than one restriction enzyme in a strain, thy are
designated in Roman numeral.
For example, Eco RI is isolated from Escherichia coli RY 13.
The final number I indicates that it is the first enzyme isolated from the
strain.
The type II restriction enzymes mostly recognize palindromic sequences to
cut the DNA.
The palindromic sequences consists of 4-6 basepairs and is bilaterally
symmetrical.
The base sequence in one strand is the same in the other strand while
reading in reverse direction.
An axis or line cuts the palindromic sequence into two identical halves,
and is called axis of symmetry.
Some restriction enzymes cut at the restriction site along the axis of
symmetry while others cut it at either side of the axis of symmetry.
PLANE OF CUTTING
Some restriction enzymes cut DNAs along the axis of symmetry of the
restriction sites
They break two phosphodiester bonds, one in ether strand of the
restriction site, at the axis of symmetry.
Hence two blunt-ends are formed. Eg. Bal I.
Several restriction enzymes cut one strand at left side of the axis of
symmetry and the other strand at the right side of the axis.
Then they break hydrogen bonds between basepairs lying between the
two cut-sites.
As a result, DNA fragments with single-stranded extensions are formed.
The single stranded extensions are called cohesive ends or stick ends.
The sticky ends of DNA fragments produced by a restriction enzyme are
complementary to each other.
The 5/position of cut end has a phosphate group and the 3/ position has
an OH- group. Egs. EcoR I, Ban HI, etc.
USES
1. Restriction enzymes are used to cut a source DNA into small fragments
for the isolation of a desired gene to be cloned.
2. They are used to cutout unwanted sequences from natural vector DNAs to
construct active vectors.
3. They are used to cut the vector DNAs at well defined sites for cloning
purpose
4. They are used to cut a large DNA into small fragments for nucleotide
sequencing
5. They are used to construct restriction map of DNAs
6. They are used to cut DNAs to determine variant sequences among the
DNAs of closely related individuals by restriction fragment length
polymorphism (RFLP).
DNA LIGASES
1. DNA ligase is an enzyme htat joins the ends of two duplex DNAs to
make a long DNA.
2. This process is called ligation.
3. It cannot add any nucleotide to a gap in the DNA.
4. It seals the neck by establishing a covalent boan between 5/
phosphate group and 3/ - OH group at the nick.
5. The bond is called phosphodiester bond.
6. This enzyme never seals the nick.
7. If there is no 5/ - phosphate group or if one or more nucleotides are
missing.
8. DNA ligase isolated form E. coli requires ATP and NAD for enzyme
activity.
9. However,DNA ligase of lambda T4 phage requires ATP alone to catalyse
the ligation.
10. This enzyme is called T4 DNA ligase.
11. It is 68000 daltons in molecular weight.
12. It has the ability to join cohesive and blunt ended DNA
fragments.
13. So it is being used in genetic engineering to join blunt ended
DNAs.
USES:
1. DNA ligase is used to join a vector DNA and a target DNA to contruct
recombinant DNA.
2. It is used to join DNA fragments of different organisms for making
vectors with desired characters.
3. It is used to add linker
4. It is used to join oligonucleotides together in the chemical synthesis of
DNA by ligase chain reaction (LCR).
RESTRICTION ENDONUCLEASES
The nuclease enzyme that cuts the DNA at a unique sequence,
is called restriction endonucleases
They cut the DNA in a non-terminal(end) region. Restriction
endonucleases are used to generate rejoinable DNA fragments
They are also known as molecular knives, molecular scissors,
restriction enzymes or molecular scalpels.
The sequence recognized by the restriction enzyme to cut the
DNA is called restriction site, restriction endonucleases site or
recognition site. The recognition site consists of 4-8 base
pairs.
The enzyme breaks two phosphodiester bonds, one in either
stand of the duplex DNA to cut the DNA. The 3’ cut end has a
free OH group and the 5’ cut end has phosphate group.
Some restriction enzymes recognize palindromic sequence to
cut DNAs, but some other recognize non palindromic
sequences.
The genome of an organism has several restriction sites for one
restriction enzyme.
The distance between two adjacent restriction sites varies
greatly.
So a restriction enzyme produces several DNA fragments of
different lengths while cutting the DNA.
TYPES OF RESTRICTION ENZYMES
The restriction endonucleases are grouped into three types.
Thry are
Type I restriction endonucleases
Type II restriction endonucleases
Type III restriction endonucleases
The type I and type III restriction enzymes recognize specific
sequence in the duplex DNA but cut the DNA far away from the
recognition sites.
So they are not useful for genetic engineering.
The type II restriction endonucleases recognize specific sites
and cut the DNA at the recognized sites.
So they are of much use in genetic engineering. Eg. EcoRI. Hind
III, etc.
S.No. Type I Restriction endonucleases
Type II restriction endonucleases
1 The enzyme is made up of three
non-identical sub-units
The enzyme is made up of
two identical sub-units
2 Molecular weight is 400000
fslyond
Molecular weight ranges
from 20000 to 00000 daltons.
3 The enzyme has both
endonucleases activity and
methylase activity.
Restriction activity alone.
4 The site of cutting is 1000 nucleotides away from the
recognition site.
The ite of cutting is the same recognition site.
5 The sequence of cutting is non-
specific.
The sequence of cutting is
specific
6 The enzymes protect DNAs by methylation.
No methylation activity.
7 ATP Mg2 adenosyl methionine
are for activation.
Mg2 alone is required for
activation.
TYPE III RESTRICTION ENDONUCLEASES:
A type II restriction endonucleases recognizes a specific
sequence in the duplex DNA and cuts the DNA at the
recognized sequence.
So the cutting is sequence specific.
The enzyme consists of two identical sub-units and its
molecular weight ranges from 20000 to 100000 daltons.
It requires Mg2 as co-factor for the enzyme activity.
Eco RI, Hind III, Mbo I, etc. are examples for type II restriction
enzymes.
At present, about 350 type II restriction endonucleases are
isolated from various bacterial strains.
They are named using the first letter of the genus name, the
first two letters of the species name and the first letter of the
strain from which the enzyme was isolated.
If there are more than one restriction enzyme in a strain, thy
are designated in Roman numeral.
For example, Eco RI is isolated from Escherichia coli RY 13.
The final number I indicates that it is the first enzyme isolated
from the strain.
The type II restriction enzymes mostly recognize palindromic
sequences to cut the DNA.
The palindromic sequences consists of 4-6 basepairs and is
bilaterally symmetrical.
The base sequence in one strand is the same in the other
strand while reading in reverse direction.
An axis or line cuts the palindromic sequence into two identical
halves, and is called axis of symmetry.
Some restriction enzymes cut at the restriction site along the
axis of symmetry while others cut it at either side of the axis of
symmetry.
PLANE OF CUTTING
Some restriction enzymes cut DNAs along the axis of symmetry
of the restriction sites
They break two phosphodiester bonds, one in ether strand of
the restriction site, at the axis of symmetry.
Hence two blunt-ends are formed. Eg. Bal I.
Several restriction enzymes cut one strand at left side of the
axis of symmetry and the other strand at the right side of the
axis.
Then they break hydrogen bonds between basepairs lying
between the two cut-sites.
As a result, DNA fragments with single-stranded extensions are
formed.
The single stranded extensions are called cohesive ends or
stick ends.
The sticky ends of DNA fragments produced by a restriction
enzyme are complementary to each other.
The 5/position of cut end has a phosphate group and the 3/
position has an OH- group. Egs. EcoR I, Ban HI, etc.
USES
7. Restriction enzymes are used to cut a source DNA into small
fragments for the isolation of a desired gene to be cloned.
8. They are used to cutout unwanted sequences from natural
vector DNAs to construct active vectors.
9. They are used to cut the vector DNAs at well defined sites for
cloning purpose
10. They are used to cut a large DNA into small fragments for
nucleotide sequencing
11. They are used to construct restriction map of DNAs
12. They are used to cut DNAs to determine variant
sequences among the DNAs of closely related individuals by
restriction fragment length polymorphism (RFLP).
PLASMIDS
Plasmids are small, circular, double-stranded, extrachromosomal DNAs
present in bacterial cells.
They are inherited sharply without the influence of chromosomal DNA.
They replicate independently due to the presence of an origin of
replication.
The plasmids are 1kbp in size and have limited number of genes.
Most bacteria contain more than one copy of each plasmid.
The number of copies of a plasmid present in a cell is called copy number.
The copy number of plasmids usually varies from 1 to 50.
However, it can be further increased by treating the bacterial culture with
chloramphenical (an inhibitor of protein synthesis).
The genes for antibiotics resistance, nitrogen fixation. Nodulation.
Environmental stresses, etc. occur in plasmid DNAs.
The antibiotics-resistance in plasmids can be used as genetic marker to
identify the strains containing the plasmids.
Some plasmids code for some secondary metabolites. Some plasmids,
under certain conditions, integrate into the chromosomal DNA of the
bacterium.
Such plasmids are called episomes ( F+ plasmid is male F—plasmids is
female ).
The integrated plasmid replicates along with the chromosomal DNA. Eg. F-
plasmid.
The eukaryotes, except yeasts, do not have plasmids.
The yeast Saccharomyces cererisiae contains Yep (Yeast episomal plasmid
or 2-mocron plasmid).
Yip ( Yeast integrating plasmid) and ARS ( Automatically replicating
sequence) in the cells.
PLASMID pBR322
Plasmid pBR322 is an artificial plasmid.
It is a gene cloning vector for E.coli.
It was constructed from two plasmids pSC101 and ColEI and a transposon
Tn3.
In the Plasmid pBR322
P indicates that it is a plasmid
BR indicates the names of workers F.Bolivar and Rodriguez, who created
the plasmid
322 is the specific number to distinguish the plasmid from others.
Plasmid pBR322 is a circular, double-standed plasmid DNA.
It consistes of 4363 basepairs.
The plasmid had 528 restriction sites for 66 restriction enzymes.
Among these 20 restriction enzymes cut it at unique restriction sites.
The pBR322 has two selectable gene markers – tetracycline resistance
gene (Tetr) and ampicillin resistancegene (Ampr) enzymes.
If a gene is inserted into any of these restriction sites the tetracycline
resistance gene becomes inactive.
The Ampr gene has unique sites for three restriction enzymes.
If a gene is inserted into any one of these unique sites, the ampicillin
resistance gene become inactive.
The Ampr gene has unique sites for three restriction enzymes. If a gene is
inserted into any one of these unique sites, the ampicillin resistance gene
becomes inactive.
The sequences other that Tetr gene and Ampr gene have unique sites for
11 restriction enzymes.
There is no insertional inactivation when a gene is inserted into any one
of these sites.
The ampicillin resistance determinant is the derivative of transposon Tn3
derived form RSF2124.
The tetracycline resistance determinant is a derivative of pSC101 derived
from R-65 plasmid.
The remaining sequence is the derivative of a ColEI derivative pMBI.
ADVANTAGES OF pBR322
pBR322 is a small plasmid consisting of 4363 basepairs
The copy number of pBR322 is 15. It can be increased upto 3000 by
adding chloromphenical to the bacterial culture.
Bacterial cells can uptake DNAs of 15 kbp size from the culture. But
pBR322 is only 4.4 kbp in size. So it can carry relatively large DNA
segments of 5-10 kbp.
pBR322 has two selectable gene markers (Tetr)and (Ampr ) for the
selection of recombinants by insertional inactivation method.
The regulation and expression of a gene inserted into the plasmid is good
pBR322 is used as a base plasmid for the invitro construction of derived
plasmid vectors such as pUC8, pUC9, pUC10, etc. and cosmids.
USES OF PLASMID pBR322
pBR322 is being used to introduce desired genes into E.coli cells eg.
Somatostatin gene of man is introduced into E.coli through pBR322.
Phagemids (OR) Phasmids:
A phagemid is a hybrid vector that has origin of replication from a
plasmid and a phage DNA.
It is constructed by inserting anlinearised plasmid DNA into a cleaved
DNA. This process is generally known as lifting the plasmid.
The DNA serves as a site for homologous recombination with
chromosomal DNA of E.coil.
Besides this, it helops for in vivo multiplication of phage particles that
have recombinant phagemids.
The plasmid portion is responsible for the independent existence of
the phagemid as plasmid in E.coil. It may be released free in E.coil. eg.
ZAP.
ZAP consists of structural genes, Ampgene Lac Z gene, T3 RNA
polymerase promoter, MCS, T7 RNA polymerase promoter, and
attachment site.
Recombinant ZAP is packaged into phage head and the recombinants
phages are allowed to infect E.coil along with a helper virus M13.
As a result the recombinant ZAP enter the E.coil and behaves like a
plasmid.
Advantages of phagemids:
1. Phagemids can be maintained as plasmids or phage particles in E.coil.
2. The desired gene can be integrated into chromosomal DNA of E.coil using
phagemids.
3. Plasmid portion can be released free as a plasmid from a
recombinant phagemid after introducing it into an E.coil strain.
4. The phages having recombinant phagemids can be stroed easily for
along time.
Lambda Phage Vector
Lambda phage is a bacterial virus that infects the E.coil. It is 57 x 106
daltons in molecular weight.
The phage consists of an icosahedral head and a flexible tail lacking
contractile sheath.
The phage DNA is packed inside the head (capsid). It is capable of
integrating into genomic DNA of host cell and transmitted through cell
division.
Therefore, it is known as temperate phage.
Lambda DNA
The DNA of phage is a linear duplex DNA with cohesive single-stranded
extensions.
It consists of 48,502 basepairs and the molecular weight is 32 x 106
daltons.
In the duplex region, more number of CG pairs are found at the left side
than the right side.
AT pairs are more at the right side. The DNA has 35-57% CG and 43-65%
AT pairs.
The single stranded extensions of DNA are complementary to each
other and consist of 12 nucleotides.
They are known as complementary sites or cos ends. The free end of
the cos site has a 5/ phosphate group.
Inside the host cell, the linear DNA becomes circular due to
complementary basepairing between the cohesive ends.
Life Cycle of Phage
The phage is a temperate bacteriophage that infects E.coil.
Althought it has both lytic and lysogenic life cycles, the latter is
more common in phage.
The frequency of lytic life cycle is increased by treating the
culture with mitomycin or exposing it to X-rays.
Lysogenic life cycle:
1. The phage attaches to the cell wall of E.coil cell with the help of tail
fibre and injects its DNA into the bacterial cell.
2. The linear DNA gets circularized by complementary base pairing
between the two cos-sites.
3. The circular DNA then integrates into chromosomal DNA of E.coil by
homologous recombination.
4. Being a part of the chromosomal DNA the phage DNA is transmitted
through cell division. The phage DNA lying within the host DNA is
called prophage or provirus. The bacterium that has prophage is
known as lysogenic bacterium or lysogen.
5. The lytic function of DNA is stopped by a repressor protein. So, in this
type of life cycle, the phage DNA does not from phages. The bacterial
cell wall has not been broken down.
6. The lysogenic bacteria are immune to second attack by phage
particles.
7. Rarely the prophage DNA gets deintegrated from the chromosomal
DNA of lysogenic bacterium and becomes a circular DNA.
8. The phage DNA produces many copies by replication. It then takes
over the protein synthesizing machinery of the host bacterium to
synthesise viral proteins.
9. As a result numerous phages are produced in the bacterium. These
phages are released free by breaking of the bacterial cell wall.
10.This type of life cycle is known as lytic life cycle.
11.The DNA usually integrates into a host DNA, especially between gal
(galactose) and bio (biotin) genes.
12.During deintegration phages receive either gal gene or biogene from
the host DNA.
13.As the sequence transduced is specific, this is known as specialized
transduction or specific transduction.
_________________________________________________________
Cosmids
Cosmids is artificial plasmid containing cos-sites of DNA. It is formed
by joining ends of a linearised plasmid DNA with cos-sites of a DNA.
It is a derived vector.
The cosmid can be packaged in capsid of phage in vitro to form
recombinant phage particles.
It is linear inside the phage cap-sid.
Cosmid has an origin if replication, selectable markers and gene
cloning sites of the plasmid DNA.
They lack structural and regulatory gene of DNA.
Hence there is nolysis and integration of cosmid DNA in the host cell,
eg. Col EI cosmid, pHC 79, pWE cosmid, etc.
Advantage of Cosmids
1. Cosmids pick up relatively larger DNA fragments than the plasmids
do.
2. As cosmids pick up large DNA fragments, they are used to establish
gene libraries of lower and higher organisms.
3. Gene cloning through cosmids helps in the study of non-sense
sequences in the genome of organisms.
4. Some cosmids are constructed by joining a linearised plasmid DNA
with DNA fragments of PI bacteriophage that have cos-ends.
5. The PI bacteriophage has the genome of 115 Kbp. So a DNA of 85
Kbp can be packaged into the head of PI phage.
6. These cosmids help to clone large genes and gene clusters in
bacteria.
Disadvantages of Cosmids:
1. The packaging enzyme fails to pack recombinant cosmids into the phage
head, if any one of the two cos-ends is missing.
2. Sometimes more than one recombinant cosmid join together to form a
large DNA. If so, the packaging enzyme fails to pack the DNA into the
phage head.
COSMIDS
Cosmid is an artificial plasmid containing cos-sites of &DNA. It is
formed by joining ends of a linearised plasmid DNA with cos-sites of a
& DNA. It is a derived vector.
The cosmid can be packaged in capsid of phage in vitro to form
recombinant phage particles. It is linear inside the phage capsid.
Cosmid has an origin of replication selectable markers and gene
cloning sites of the plasmid DNA.
They lack structural and regulatory genes of DNA.
Hence there is no lysis and integration of cosmid DNA in the host cell.
E.g. Col El cosmid, pHc 79, pJB8, pWE cosmid, etc.
Cosmid was first constructed by Collins and Hogn in 1978.
Salient Features of Cosmids
1. Cosmid is a circular double stranded DNA.
2. It has two complementarty single stranded regions at both ends of a
plasmid DNA. The two cos-ends form a duplex by base pairing.
3. At the cos-site, 3/ end and 5/ end of the same chain do not establish
covalent bond during circularization. So a definite nick is present in each
DNA strand.
4. The nicks are retained in the plasmid for a number of generations.
5. The cosmid DNA does not code for phage proteins and host cell lysis.
6. It does not involve in multiplication of phage particles.
7. It has an origin of replication from plasmid DNA for independent
replication.
8. It has selectable marker genes and gene cloning sites of plasmid DNA.
9. The cosmid DNA is packaged within protein coat of bacteriophage to form
infective phage particles. Cos-site is a prerequisite for invitro packaging of
cosmid in phage protein coat.
10. After infection, the cosmid DNA does not integrate into chromosomal
DNA of host cell. It exists as a definite extra chromosomal DNA and
replicates independently.
Cosmid pLFR5
PLFR5 is the commonly used cloning vector suitable for
cloning large DNA fragments upto 45 K bp.
It is 6 Kbp in size. It is constructed from E.coli plasmid pBR322
and two cos-ends of DNA.
The plasmid derived portion contributes an origion of
replication (Ori) and tetracycline resistance gene (Tetr).
There is a multiple cloning site (MCS) between the origin of
replication and cos-site.
A foreign DNA of upto 45 kbp is inserted into the MCS of
pLFR5 and the rDNA is packaged into bacteriophage head in
vitro.
The phage thus formed delivers the DNA into E.coli while
infecting the cell.
Simian Virus 40 (SV40)
SV40 is a small virus that infects monkeys (simian). It causes
cytoplasmic vacuolation in infected cells and hence it is known as
Simian Vacuolating Virus 40.
SV40 is an oncogenic virus. It has been used as a gene cloning vector
for mammalian cells.
SV40 consists of an outer protein coat called capsid and inner DNA and
histone core. The capsid is icosahedral in shape.
The DNA is circular and double stranded. It is about 5.2 kbp in size. It is
found associated with his tone proteins such as H4, H2a, H2b and H3 in
the form of a minichromosome.
The DNA of SV40 contains an origin of replication, a set of genes called
early genes and another set of genes called late genes.
The early genes are seen in one strand and the late genes are present
in the other strand. The early genes produce two mRNAs for tumour
antigens.
Life Cycle of SV40:
SV40 follows two types of life cycles. When it infects permissive cells
such as cell lines of African green monkey, it multiplies within the cell
and brings about host cell lysis. It is called lytic infection.
The lytic infection takes place in three phases:
The first phase lasts for 8 hours. During this phase, SV40, loses its
protein coat in the cytoplasm, and its DNA moves to the nucleus.
The second phase is the early phase and lasts for 4 hours. During this
phase mRNA synthesis, DNA replication, etc. take place.
The third phase is the late phase which lasts for 36 hours. During this
phase synthesis of virus capsid, assembly of capsid and maturation of
SV40 particles take place.
At the end of this phase viral particles come out of the cell by breaking
the cell membrane.
When SV40 infects non-permissive cells, it integrates its DNA into
genome of the host cell.
The virus does not multiply in the cell.
Therefore, there is no cell lysis. However, the transformed cells get
oncogenic property.
The SV40 sequence may get amplified and rearranged in the cells.
SV40 As Vector:
There is no long non-essential sequence in SV40 DNA for gene cloning.
So the foreign DNA is inserted into SV40 DNA either by replacing the
early genes or late genes.
Deletion of genes make the vector replication defective one. The rDNA
is package in protein coat of SV40 to have recombinant SV40 particles.
The host cells are co-infected with recombinant SV40 and another
helper virus.
The helper virus supplies the functional copies of missing genes in the
recombinant DNA.
This will help for the in vivo replication of recombinant DNA and
multiplication of SV40 in the host cell.
Usually host cells are lysed at the end of infection so that it is difficult
to get transformed mammalian cells using this vector.
Gluzman (1981) developed COS cells by gene cloning and recombinant
viruses were allowed to infect the host cells.
As COS cells contain early genes, it complements the defect in
recombinant DNA.
So there is no need for helper virus for gene transfer. Here virus
multiplies in the cell and breaks down the host cell.
Now DNA fragment of SV40 containing origin of replication is linked
with an E.coli plasmid vector to construct a shuttle vector.
When foreign DNA is inserted into such vector, the rDNA can replicate
in the mammalian cells. Further it does not cause host cell lysis.
This technology is of much use for gene transfer into mammalian cells.
UNIT – III
I-3-RECOMBINANT DNA (rDNA) TECHNOLOGY
Gene transfer refers to the invivoproduction of multiple copies of a
desired gene.
It involves the invitro construction of rDNA and amplification of the rDNA
in a bacterium.
Gene transfer involves many complex techniques. Gene transfer is
discussed here with reference to insulin gene. It involves the following
steps.
i. Preparation of desired (insulin) gene
ii. Preparation of plasmid (pBR322)
iii. Insertion of desired (insulin) gene into plasmid (pBR322)
iv. Introduction of rDNA into E.coli.
v. Selection of recombinants.
PREPARATION OF DESIRED (INSULIN) GENE
Insulin gene can be obtained from genomic DNA.
The genomic DNA of man (the source organism) contains insulin gene
along with numerous other gene. The cell DNA is isolated in the following
way.
Human cells in a tube is treated with the detergent SDS(sodium
dodecylsulphate) to disrupt the cell membrane
The cell lysate is treated with proteinase k to digest cellular proteins and
histones bound to the DNA.
It is then treated with phenol-chloroform mixture to precipitate proteins.
The protein precipitate is centrifuged out. The supernatant is treated
with ribonuclease to digest RNAs.
Then the supernatant is treated with acetate-ethanol solution and stirred
well with a glass rod. A white fibrous precipitate of DNA develops on the
glass rod
The precipitate is washed with TE(Tris-E) buffer and then with ESAM
solution.
The DNA precipitate is finally suspended in TE buffer and stored at -200C
for future use. The cell DNA has insulin gene atleast in one locus.
PREPARATION OF PLASMID (pBR322)
The plasmid pBR322 has been a popular vector for gene cloning in E.coli.
It is an artificial plasmid
It is constructed from plasmid pSC101, pMB1and RSF2124.
When pBR322 is required. It is isolated from the E.coli cells. It involves
the following steps.
The bacterial cell wall is ruptured by treating with lysozyme and EDTA.
The cell is lysed with SLS.
The cell wall debris and proteins are centrifuged out
The supernatant is fractionated by using cesium chloride density gradient
centrifugation. Plasmids go to the lower layer.
The plasmids are taken from the centrifuge tube and used for gene
transfer.
INSERTION OF INSULIN GENE INTO pBR322.
The cell DNA is cut with a restriction enzyme to get small DNA fragments.
Among these fragments, one contains insulin gene.
However, we do not know in which fragment insulin gene resides.
Therefore, the DNA fragments are separated from one another by
electrophoresis.
Each fraction is then cloned separately.
The pBR322 is cut with a restriction enzyme that has unique site at its
antibiotics resistance gene and that was used to cut the cell DNA
The lineariszed plasmid DNA is mixed with a fraction of cell DNA in 1:3
ratio. The cohesive ends of the plasmid DNA and the cell DNA fragment
anneal by complementary base pairing
The nick between the two DNAs is sealed by using DNA ligase.
The hybrid DNA thus formed is called recombinant DNA.
In such a way rDNAs are constructed with DNA fragments of each and
every fraction of cell DNA.
Of these different rDNAs, atleast one type contains insulin gene.
INTRODUCTION OF rDNA INTO E.coli
Recombinant pBR322 is introduced into E.coli cell by bacterial
transformation.
The rDNA is added to an E.coli culture and the culture is treated with
50mM calcium chloride(CaCl2) solution at room temperature.
CaCl2 adheres the rDNAs onto the surface of E.coli cells and modifies the
bacterial cell wall to intake the rDNAs.
The bacterial culture is then heated gently to 420C that stimulates the
E.coli cells to intake the rDNAs.
As a result E.coli cells become recombinants.
SELECTION OF RECOMBINANTS
Recombinants are cells or organisms harbouring recombinant DNAs.
They are selected from non recombinant cells (wild type cells) in the
following steps.
LOSE OF ANTIBIOTICS RESISTANCE:
I. pBR322 confers resistance to ampicillin and tetracycline.
II. Here Tetr gene was inactivated by the foreign gene.
III. So the rDNA does not confer tetracycline resistance to the recombinants.
IV. After transformation, E.coli culture is plated onto an agariszed medium
containing ampicillin.
V. Then a replica plate is made onto an agariszed medium containing
ampicillin and tetracycline.
VI. These colonies of the master plate failed to form their replica in the
replica plate are recombinants.
VII. The recombinants are cultured in a suitable medium.
APPLICATIONS OF GENE TRANSFER TECHNIQUE
Gene transfer has the following applications
Gene transfer method is used to establish gene banks or DNA library of
organism
It is used to study the structure and regulation of genes in living beings
It is used to amplify the desired gene for DNA sequencing
Gene transfer provide enough amount of desired gene or gene
manipulation
In some gene transfer, the desired gene expresses its protein. In such
cases, the gene transfer is cultured in large amount to get the valuable
protein for human use. Eg. Human insulin.
In Vitro Fertilization:
The process of fertilization an egg with a sperm in a nutrient medium in the
culture plate is known as in vitro fertilization (IVF). This method is practiced in
farm animals and women having damaged fallopian tubes. It overcomes the
difficulties in fertilization in such individuals.
Unfertilized eggs are collected from the female and placed in the nutrient
solution in a culture plate. Semen is collected from a male and added into the
plate to fertilize the eggs. The culture plate is then incubated at 370C for a few
days till the fertilized eggs reach 8-16 cells stage. The globular embryo is then
implanted into the womb of the female. After a suitable period conception the
female produces a young one as usual.
Cattle Breeding:
In vitro fertilization is used in-
1. Cattle breeding
2. Breeding rare and endangered species using another species of surrogate
mother women who carries a fetus
3. Developing human test tube babies
Raymond’s Embryo Research Centre (Madhya Pradesh) has bred high yielding
calves through inferior quality cows.
Holstein – FreeianVariety cows produce a large quantity of milk but they suffer
frequent abortion. Malvi breed, a native variety of Madhya Pradesh, is poor in
milk yield but resistant to abortion.
ShyamZawarand co-workers had taken the egg form Holstein-Freeian variety
cows and fertilized in vitro with semen collected from the bull. Having attained 8-
16 celled embryo, it was implanted in the womb of the Malvibreed. The method is
known as embryo transfer technology.
Breeding Rare Species:
IVF technology is used to save rare species of animals from extinction. Many
wild animals have been bred by this technique. In a London zoo. Wild Indian bison
and guar have been bred by using Friesan cow a surrogate mother. Horse mares
were made to give birth to Britain zebra.
Test Tube Babies:
Babies raised from in vitro fertilized eggs are called test tube babies. It
stimulates the development of several follicles.
A small slit is made just below her noval and an instrument called laparoscope
is inserted into the slit. The laparoscope has a light source, a lense system and a
micropipette. The surface of the overy is viewed throught the lense system for
searching mature follicles and the mature follicles are taken out through the
micropipette.
The eggs (follicles) are inoculated into a nutrient medium containing bovine
serum albumin and pyruvate. Semen is collected from the woman’s husband or
any other man and added into the medium. The culture plate is incubated at 360C
for a few days to notice the fertilized eggs.
At the 8-16 cell stage, an active embryo is implanted into the uterus of the
woman by using a micropipette. In 38 weeks she will produce a baby.
This method helps immensely the infertile woman with badly damaged
fallopian tubes.
Xerox Animals:
The offspring which are the exact copies of their parents are called Xerox
animals or copy animal or clones. The method of making Xerox animals is referred
to as animal cloning. For the first time Xerox frogs were made by Robert Bricks in
1952. Later it has been practiced in fishes, poultry, rats, pigs, goats and cows.
Ian Wilmotand his co-workers at Roslin institute in Scoland for the first time
succeeded in cloning a mammal in 1997. They created a cloning goat named
Dolly. Dolly is the Xerox copy of the mother.
Some mature somatic cells were taken from the udder of a donor ewe having
whitish face. They were placed in a nutrient solution in a Petri dish and incubated
at cold temperature (less than 50C)
Then an ewe having brownish face was treated with gonadotrophin in
injection to stimulate the development of eggs. Mature eggs were taken from the
ewe using a laparoscope and placed in a nutrient solution. From the mature egg
cell, the haploid nucleus was sucked out using a micropipette under a microscope.
Later, a diploid nucleus was taken from the somatic cell and introduced into
the enucleated egg cell. The egg cell was then allowed to grown in the medium till
it reached 8-16 cells, stage. That cell mass was implanted into the uterus of the
brown faced ewe.
At the end of the 6th month the ewe delivered a white faced female goat . Dolly
is the exact copy of the white faced adult ewe.
This method can also be adopted for human being and is called human
cloning. However, it has not been practiced so far because of some social and
ethical issues.
Embryo Splitting:
The embryo of monkey at 8- celled stage is split into two partially separated
units using a nylon thread. This embryo when implanted into uterus of a monkey,
produces identical twins. This technique is known as embryo splitting.
UNIT – IV
DNA Fingerprinting
Distinguishing the individuals according to their DNA prints pattern is called
DNA fingerprinting. It is also called DNA profiling or molecular fingerprinting. It is
used to assess whether an individual has genetic relationship with supposed
individuals or not.
Methodology
DNA fingerprinting involves the following steps:
1. Isolation of cell DNA
2. Restriction digestion
3. Gel electrophoresis
4. Southern blotting
5. Selection of DNA probe
6. Filter hybridization
7. Autoradiography
8. Analysis of DNA print pattern
Isolation of cell DNA:
The cell DNA is obtained from blood or semen clotted on cloths, vaginal
swabs taken from rape victims, hairs or fresh cells or blood. DNA is isolated from
the source material and the suspected individuals.
Restriction Digestion:
The cell DNAs are separately cut with a restriction enzyme that cuts on either
side of a mini-satellite. As a result minisatellites get released from the cell DNA.
Gel Electrophoresis:
Each DNA restriction digest of the resource persons and source material, is
poured into a well of an electrophoresis gel and electrophoresed. The
minisatellites separate according to their lengths.
Southern Blotting and packing:
The separated DNA fragments are transferred to a nylon membrane or a
nitrocellulose filter paper by placing it oyer the gel. This process is called southern
blotting.
The nitrocellulose filter having DNA fragments is dried in between dry filter
papers at high papers at high temperature. This process is called packing.
Selection of DNA Probe:
In the USA, DNA probes constructed by Alec Jeffreys are used for DNA
fingerprinting. However, these probes are not available to India. Dr.Lalji Singh at
the Centre for cellular and molecular biology in Hyderabad, has developed a DNA
probe from minisatellite DNA of banded krait (Bungarus fasciatus) and named it
BK m probe. The BKm probe is being used for DNA fingerprinting in India.
Filter Hybridization:
The nitrocellulose filter is placed in an alkali solution to denature the duplex
DNA fragments. Then it is placed in the hybridization solution containing the
probe DNA. The probe DNA binds with appropriate minisatellites and forms
duplex DNAs.
Autoradiography:
After hybridization the filter is washed with a wash solution to remove
unbound probes. Then an X-ray film is placed over the filter for about 3 hours.
The radioactivity of the probes makes dark spots on the x-ray film. Therefore, an
irregular ladder of dark spots develops on the x-ray film, for each and every DNA
sample. Each ladder represents a DNA print of an individual. All DNA prints on the
x-ray film together constitute a DNA fingerprint pattern.
Analysis of DNA Fingerprint pattern:
In the case of rape and murder, all bands of seminal DNA or hairs-DNA should
match perfectly with DNA print of the accused. If the match is 100%, the
individual is found to be responsible for the victim. All bands of source material
never match with the bands of DNA print of any person who didn’t involve in the
case.
In the Fig.19.1 all bands of DNA print obtained from vaginal swab match
with the bands of DNA print of person A with 100% certainty. The person B and
C’s DNA prints never match with the DNA print of X. So A is believed to be the
accused.
For determining the parentage of a child, 50% of bands in the child’s DNA print
should match with those of the father’s DNA print and the rest of the bands
should match with those of the mother’s DNA print. This is because of the fact
that half of the DNA of the child came from the father and the other half of the
DNA came from the mother. If some of the bands in the child’s DNA print do not
correspond with either the alleged father or alleged mother, then it is certain that
they are not its real parents.
Applications of DNA Fingerprinting
DNA fingerprinting has been used to settle disputed parentage, murder cases,
distinguish rapists and to analyse the genetics of various organisms.
To Settle Disputed Parentage:
In some abnormal cases, courts cannot decide who is the real father or mother
or both of a child. So it is difficult to settle the case. DNA fingerprinting is used to
settle the case
Example-1:
In 1984, a Ghanaian family with a mother, two sons and two daughters, went
to enter Britain. The immigration authorities refused the entry of one boy into the
Britain, as they thought that the boy was not her son. But the woman said that
the boy was his own son. Studies on enzymes, antiserum and erythrocytes didn’t
help to solve the problem.
A.Jeffreys made two DNA fingerprint patterns form DNAs isolated from
all members of the family using two multilocus probes. They identified 25
maternal specific bands out of 61 bands in the boy’s DNA print. Hence they
concluded that the boy was her own son. Finally the family was allowed to enter
the Britain.
Example-2:
The High Court of India, Hyderabad, faced a tricky case in 1989. A woman
named Kanniammal and her husband Adiveerarama pandian reported to the
police that young daughter lakshmim was missing from their residence. The police
couldn’t till 15th march, 1989, about 10 months after the incident.
On that particular day, pandian saw a girl about 5 years old, having similar
appearance to his own daughter lakshmi. But the girl was in the company of a
couple, Perumal and his wife kaliammal. Pandian believed that perumal and his
wife had kidnapped his daughter. He demended hat his daughter to be returned
to him. Perumal and Kaliammal obviously refused it. Hence the case reached the
court.
The magistrate at that court couldn’t order to cut the baby into two halves so
that each couple could keep one half of the baby. He requested Dr.Lalji Singh of
CCMB. Hyderabad to assist the case. Lalji Singh developed two DNA print patterns
separately with the two couples and the girl. The DNA fingerprint analysis is given
in the figure 19-2.
Out of eight bands in the DNA print of the girl, 4 matched with DNA print of
perumal and the other four matched with DNA print of Kaliammal. No one of the
bands match with the bands in DNA print of neither pandian nor kanniammal. On
the basis of DNA fingerprinting evidence, the court ordered to keep the baby with
perumal and kaliammal. They are its real parents.
Fig.19.2: Analysis of DNA fingerprints in paternityesting. A DNA print pattern
of perumal (p) – Lakshmi (L) – Kaliammal (Kal). B-DNA print pattern of
Adiveerarama pandian (AP) – Lakshmi (L) – Kanniammal (Kan).
To Settle Murder Cases:
DNA fingerprinting helps the police and law courts to little some
complicated murder cases.
Example- 1:
In 1995, a man found a piece of index anger with nail in tandoori provided
by Bagia Restaurant and be informed it to the police. There was no clue to find
out the Actium and accused. Finally, DNA finger printing was made from DNAs
isolated from tissue recovered from the tandoori and hairs of doubtful persons
who were missing recently, It confirmed that the index finger was Naina Shani’s
one. Then the culprit sushil Sharma was arrested on the basis of DNA
fingerprinting evidence.
Example – 2:
In Canada, one man was murdered in 1997. The murderer never left any
fingerprint or blood, except a few cat’s hairs on the coat of the dead body. DNA
print to the cat’s hairs matched with that of hairs taken from a cat of a suspected
person. So he was convicted for the case and punished.
Example -3:
By using DNA fingerprinting, a man charged for many years with a murder
case, was shown to be innocent and left free from the jail. It happened in the
Britain in 1987.
3. To Distinguish Rapist:
If a lady was raped by a man in a dark night and she was unable to find out the
man at that time. DNA fingerprinting helps to distinguish the accused from men
arrested doubtfully.
DNA fingerprint pattern is made from DNAs taken from vaginal swab and
semen or blood of doubtful persons. If the DNA print of the vaginal swab matches
with DNA print of any one man with 100% certainty, it is confirmed that the
particular man is accused for the rape.
4.Pedigree Analysis:
DNA fingerprinting is used in pedigree analysis in cat, dog, horses and man.
5. Migration pattern:
It is used to assess the pattern of migration of ancient populations.
6. Genetic Analysis:
It is also employed in genetic analysis of various strains of agricultural crops
and animals.
DNA SEQUENCING
The human genome project is a multinational research project to determine
the genomic structure of man (Homo sapiens). It is aiming at sequencing all DNAs
of man and at determining the location of various genes in the DNAs. Many
Government and private sectors have been taking part in the on-going project.
The genome project was initiated in 1988. It is under the international
administration of the ‘HUMAN GENOME ORGANIZATION’(HUGO).It is funded by the
DEPARTMENT OF ENERGY (DOE) AND NATIONAL INSTITUTES OF HEALTH (NIH) IN the USA, the
EUROPEAN COMMISSION (EC)and Britain’s Welcome Trust. Many puplics have also
been donating for this scientific investigation.
The research works in the project have been conducted in research
laboratories in six nations. The most important among them are the National
Human Genome Research Institute (USA), Sanger Centre (England) and Celera
Genomics. 12000 base pairs of human DNA have been sequenced in every
minute. It is expected that the project will be completed in the year 2005.
ADOPTED METHODOLOGY
A somatic cell of human being contains 23 pairs of chromosomes. Each
chromosome is composed of a long double stranded DNA and his tone protein. It
is estimated that there are 3.2 million basepairs in the DNA of all these
chromosomes. Among them 2 million basepairs were already sequenced. The
general methodology of the genome project is outlined below:
OBTAINING DNA:
A cell culture containing a human cell line is homogenized in sucrose solution
using a pestle. The homogenate so obtained is subjected to differential
centrifugation to get a chromosomal fraction. This fraction is added with a lysis
buffer and again centrifuged using an ultracentrifuge to separate different
chromosomes according to their sizes. Thus human chromosomes 1-23 are
isolated and numbered properly.
In the chromosomes, DNA is wrapped around his-tone proteins in the form
of small balls. These DNA wrapped units are called nucleosomes. Each
chromosome, say for example chromosome 1, is suspended in a solution
containing Tris-Hcl, EDTA and NaCl and stored at -200C. This treatment releases
histones from the nucleosomes, releasing the DNA free. The DNA is isolated from
it by Cesium chloride density gradient centrifugation. Each DNA is about 5 feet
long.
Preparation of DNA for Study:
The long DNA of a chromosome is cut into small pieces of 5000-10,000
nucleotides using a restriction enzyme that cuts the DNA at long distances. The
individual pieces of DNA are separated by agarose gel electrophoresis on the basis
of restriction fragment length polymorphism (RFLP). Each and every DNA
fragment so obtained is placed in a vial and stored at 200c for future use.
One DNA fragment is inserted into a plasmid DNA to construct a rDNA
fragment. Consequently billions of copies of that DNA fragments are made. In this
way each and every DNA fragment is amplified.
Sequencing and Analysis:
The amplified rDNA is isolated from the bacterial cells and the target DNA is
separated. It is cut with a restriction enzyme to generate small DNA fragment.
Fluorescent dyes visible under laser light are added to the terminal nucleotide of
each DNA fragments. The resulting DNA solution is poured into 96 tubes inside
the DNA sequencing machine. In the tubes the DNA fragments are electrophored
very fast and this can be observed by a fluorescence recorder in the gene
machine.
The bases in the overlapping segments are identified and assembled in a linear
order by using computer database. In this way all DNA fragments of a
chromosome are sequenced to recreate its original nucleotide sequence.
Such a study is conducted on all 23 chromosomes of human genome to
understand the exact genome structure of man.
Applications of Genome Project
1. Genome project provides database information of DNA sequences of man.
Biotechnology based companies may use the information to manufacture
human proteins which are of much use in the human disease treatments.
2. It helps to detect genetic disorders in man and their inheritance. 289
genetic diseases have been known.
a. Chromosome 1 : GBA gene - Gaucher disease
HPC 1 gene - Prostrate cancer
PS 2 (AD4) - Alzheimer’s disease
b. Chromosome 7 : GCK gene - Diabetes
CFTR - Cystic fibrosis
OB gene - Obesity
c. Chromosome Y : SRY (TDF) - Testis differentiation factor
3. A proper remedial gene can be choosen and administered to treat genetic
disease.
4. The action of harmful genes is blocked by introducing an antisense gene to
step the genetic disease.
5. The American company, Incyte Genomic has manufactured gene chip with
10,000 gene kits. This chip can be used to detect genetic diseases,
infectious diseases, oncogenes, parasitic worms etc, at once during clinical
diagnosis. The diagnosis is very fast: it will be over within 3hrs.
6. Genomic project will help to understand what the real bases of human life
are? Walter Gilbert a professor of Harvard University-says ‘What is to be a
man will come to our knowledge when the Genome project is completed’.
7. By matching the human genome with the genome of Drosophila. Scientists
conclude that this fly has remedial genes for 177 genetic diseases in man.
So the remedy is always around us.
8. The information about the genome will be utilized to design babies with
many superior characters such as skill, strength and free of genetic
disorders.
DNA Microarray
Microarray technology has provided HTPS methods for diagnosis and screening
of different genetic disorders and pathogenic diseases. Microarrays can be
classified on the basis of materials arrayed upon them. They have been
constructed using DNA, cDNA, proteins, antibodies, carbohydrates, cells and
tissues. Several such microarrays are commercially available for ready use.
Nanotechnology makes use of fabrication tools for the manufacture of
microarrays and biochips.
Fluorescent chromophores, quantum dots, nanoparticles, cantilevers and
nanowires are employed in microarrays for sensitive detection of compounds in
the analytes.
DNA Microarray
Ordered arrangement of DNA probes on silicon surface is called DNA microarray
or gene chip. In DNA chips, 16,000 different DNAs are immobilized in an area of
12cm2. Latest developments in DNA microarray have enabled us to immobilize
about 16,000 different DNas in 1cm2 area of silicon surface. These microarrays are
called high density arrays. By using microarrays, thousands of diseases or
complementary strands can be detected at a time. After washing, the
microarraychips can be reused for further tests.
Genomic DNA is isolated and cut into small pieces using a restriction enzyme.
Using gene probe PCR, the selective sequence is amplified in vitro. The PCR
product is separated using electrophoresis and then labeled with a fluorescent
compound. The gene chip is kept dipped in the dye-labelled DNA solution. After
washing the gene chip, it is scanned using laser setup. The dot of microarray that
is hybridized with the labeled probe fluoresces green or red light depending on
the dye. From the specific dot giving positive result, we can get complete details
of the gene by means of insilico analysis.
Genomic DNA
Restriciton
enzyme
Restriction digest Gene chip
PCR Labelling with
Ampli fied DNA fluorescent dye
Positive dot Gene chip
Uses of DNA Microarray:
DNA microarray is used to detect gene expression by analyzing
cDNAs produced from mRNAs of a cell types at different times.
It is employed in genotyping of genomes through single nucleotide
polymorphism (SNP) analysis.
With the help of species specific probes, DNA microarray is used to
identify microbes in the environment.
Gene chips are available to diagnose several pathogenic and
genetic diseases in man.
Microarray is used in the analysis of transcriptomes and proteomes.
_______________________________________________________________________________
GENE THERAPY
The treatment of genetic diseases by introducing proper genes into patient’s
cells is called gene therapy. The gene used to treat a genetic disease is called
remedial gene or remedial DNA or gene drug.
Human being suffers many diseases due to defects in their genetic makeup.
Such diseases are called genetic diseases or genetic defects. They cannot be cured
permanently using chemotherapeutic drugs. These genetic diseases are cured by
introducing proper remedial genes into patient’s body.
The remedial gene is introduced into germ cells such as egg, sperm and zygote
or somatic cells such as liver cells, skin cells and bone marrow cells. The remedial
gene may replace the function of the defective gene or block the activity of the
defective to cure the disease.
Examples: Thalassemia
Leukemia
Diagnosis of Genetic Diseases
There are over 2500 genetic diseases in human being. Among these, some are
more dangerous to the sufferers as they cause illness and death. Now they can be
diagnosed at early stages in the following routes:
1. Parental Screening:
Some genetic diseases have been inherited to children from parents.
Such diseases can be detected by testing parents (people) whether they
suffer genetic defects or not parental DNA is hybridized with known DNA
probes to diagnose the genetic diseases in man.
Usually parental screening is used to advise people not to have children
suffering genetic diseases.
2. Antenatal Screening:
Antenatal screening is the diagnosis of genetic disorders in fetus while it is
in mother’s womb. It is also called pre-natal genetic screening. The foetal
DNA is obtained from cells in liquor amnii or from cells in chorionic villi at 8-
16 weeks of pregnancy. Then it is hybridized with known probes.
DNA probes are available to detect the following genetic disorders-
Mutation in Dominant Mutation in Recessive
Genes in Autosomes genes in Autosomes
Hypercholesterolemia Cystic fibrosis
Polycystic kidney disease Phenylketonuria
Huntington’s cholera Sickle cell anemia
Neurofibromatosis Thalassaemias, etc.
Mutation in X-linked
Recessive genes
Haemophilia A
Haemophilia B
Duschenne muscular dystrophy, etc.
Postnatal Screening:
Testing of newborn children for genetic disease is known as postnatal
screening. DNA hybridization with known probes is used for this purpose. The
American Academy of pediatrics (AAP) in 1989 has recognized the following
methods to diagnose genetic defects in newborn children:
I. Enzyme assay method (to detect Biotinidase and Muscular dystrophy).
II. Enzyme immunoassay (to detect congenital adernal hyperplasia).
III. Radioimmunoassay (to detect congenital hypothyroidism).
IV. Immunoreactive trypsin (to detect cystic fibrosis).
V. Bacterial inhibition (to detect Galactosemia Homocystiuria Maple syrup,
disease etc).
VI. Protein electrophoresis (to detect sickle cell anemia, Thalassemia, etc).
Gene Therapy Methods
There are two methods in gene therapy. They are-
1. Germline gene therapy
2. Somatic cell gene therapy.
Germline Gene Therapy:
Treatment of genetic diseases by introducing a remedial gene into sperm, egg
or zygote is known as germline gene therapy. The gene may be introduced into
germ cells by using microinjection, biolistics or liposome fusion. The introduced
gene corrects the genetic disorder in the cells. It has been inherited to future
generations.
Rubin and Sprading 1982 have microinjected xanthine dehydrogenase (XD)
gene into eggs of Drosophila having defective XD gene. The flies developed from
the eggs are normal and produce xanthine dehydrogenase enzyme.
Neuromuscular disease, in mice is due to mutation in 427 K gene. J.S
Chamberlain 1993 microinjected a 427 K gene into a zygote of mice suffering the
disease. The remedial gene has corrected the disease in the off springs.
Somatic Cell Gene Therapy:
Treatment of genetic diseases by introducing a remedial gene into somatic cells
is called somatic cell gene therapy. The remedial gene is introduced into liver cells
spleen cells, muscle cells or blood cells.
If a remedial gene is introduced into an young embryo, that treatment is
called embryo therapy. If a gene is introduced into somatic cells of an adult
patient, it is called patient therapy.
In general somatic cell gene therapy is of 4 types-
1. Embryo therapy 2. Ex Vivo therapy 3. In Vivo therapy 4. Antisense therapy
Embryo Therapy:
Treatment of genetic disease by introducing a proper remedial gene into cells
of 2-10 days old embryo is called embryo therapy of fetal gene therapy. This
method was devised by A.Handyside et al. in 1993 to cure cystic fibrosis.
The egg and sperm were collected from patients suffering cystic fibrosis and
fertilized in vitro. After getting 8-celled embryo, one cell was taken from the
embryo for gene manipulation, Remedial gene for cystic fibrosis was inserted into
a plasmid to construct rDNA. The rDNA was introduced into the embryonic cell.
The recombinant cell was transplanted back in the embryo. The embryo delivered
a baby free from cystis fibrosis.
Ex Vivo Therapy:
In this method, some cells are taken from an appropriate organ of patient,
remedial gene is introduced into the cells, and the cells are transplanted back into
that organ. Ex vivo gene therapy is also known as transplantation or tissue
grafting.
Examples: Bone marrow transplantation Liver transplantation Kidney transplantation
Bone Marrow Transplantation:
Bone marrow is a soft tissue in the medullary cavities of bones. Immuno-
deficiency diseases are mainly concerned with the derivatives of marrow cells. So
they can be cured by bone marrow transplantation. Eg. Severe combined
immunodeficiency (SCII).
Severe combined immunodeficiency is a genetic disease in man due to
mutation in adenosine deaminase gene. Adenosine and deoxyadenosine get
accumulated in the blood. These substances are toxic to B-cells and T-cells. The
sufferer dies within 3 years after birth.
The bone marrow transplantation to treat SCID is outlined
below:
1. A small piece of bone marrow is taken form a bone of patient suffering
SCID.
2. The marrow is macerated and cultured in supplemented Hanks-Simm’s
medium.
3. Murine leukaemia virus (Mul V), a retrovirus, is allowed to infect a mouse
cell line. By reverse transcription the RNA produces duplex DNA called
Proviral DNA.
4. The proviral DNA is isolated from the mouse cells.
5. Gag, poly and env gene in the proviral DNA are deleted. A neomycin
resistance gene (Neor) was inserted between the encapsidation region and
viral replication sequence of the vector.
6. The remedial gene adenosine deaminase gene is inserted near the
encapsidation region of the vector to construct an rDNA.
7. Mouse cells in a culture are treated with the rDNA and calcium phosphate
solution. Calcium phosphate induces the cells to intake the rDNA. This is
called infesting.
8. The infested mouse cells are again infected with a helper virus. The helper
virus lacks encapsidation region. So it produces virus coat protein but its
RNA cannot be packaged into the protein coat. At the same time, the rDNA
has encapsidation region foe packaging. So mRNA produced from the rDNA
gets packaged into the protein coat to form an infective virus particle.
9. The virus particles are isolated from the infested cells and allowed to infect
T cells isolated from the marrow cells. The cells are incubated for a few
days. The virus injects rDNA into the cell: the RNA is made into duplex DNA.
10. The T cells in the culture are then treated with the antibiotic neomycin. T
cells with rRNA are resistant to neomycin.
11. The successful cells are washed with ddH2O and transfused back into
marrow of the patient’s bone. The infection of individual cells in a
suspension into the bone marrow is called transfusion.
In yet another method the T cells are growth into cell masses. The cell
mass is placed in marrow of the patient’s bone from which it was taken out
previously. This method is called autoplastic transplantation.
12. The transplanted cells produce adenosine deaminase and cure SCID
disease in the patient.
T cells have short life time so repeated transfusions may be needed (2-
7 times) to cure the disease.
Some genetic diseases that can be cured by bone marrow
transplantation are listed below:
1. A drenoleukodystrophy 11. Macroteaux-Lamy syndrome
2. Chediak – Higachl dyndrome 12. Metachromatic laukodystrophy
3. Chronic granulomatous
Disease 13. Osteopetrosis
4. Fanconi anemia 14. Purine nucleotide hosphorylase
5. Gaucher disease deficiency
6. gPL-115 deficiency 15. Reticular dysgenesis
7. Granulocyte actin deficiency 16. Sanfilippo syndrome
8. Hunter disease 17. Sickle cell anemia
9. Hurler syndrome 18. Thalassemia
10. Infantile agranulo- cytosis 19. Wiskott – Aldrich syndrome
20. X- linked agammagl obulinemia 21. Melanoma
Liver Transplantation:
Ex vivo gene therapy using liver cells is called liver transplantation. Diseases such
as familial hypercholesterolemia, hyperammonemia, etc. can be corrected by liver
transplantation.
Familial hypercholesterolemia is characterized by high cholesterol level in
blood and coronary artery disease. It is due to mutation in low density lipoprotein
receptor gene (LDLRgene). The LDLR is produced in liver cells.
Some liver cells are taken from the patient and grown in vitro. LDLR gene is
inserted into a retroviral DNA in a plasmid to construct an rDNA. The rDNA is
transfested into mouse cells using calcium phosphate solution. The mouse cells
are then infected with a helper retrovirus to from infective virus particles.
The virus particles are then allowed to infect the liver cells. After proper
screening. The liver cells containing the rDNA are transfused into the liver of the
patient. The remedial LDLR gene corrects the genetic disorder in the patient.
In Vivo Gene Therapy:
The direct delivery of a remedial gene construct into proper organ of a patient
to correct the genetic defect is called in vivo gene therapy. It is less time
consuming and easy to perform. Duchenne muscular dystrophy, Alizheimer’s
disease, brain tumour, etc. are being treated in this way. Adenovirus and herpes
simplex virus are used as gene delivery systems.
1. DNA is isolated from an adenovirus to construct a recombinant virus.
2. A segment of DNA having E1A, E1B and E3 genes is deleted from the
viral DNA.’
3. Dystrophin gene is inserted at the deleted site of the victor DNA to
construct an rDNA.
4. The rDNA is transfected into a mouse cell line using calcium
phosphate.
5. The mouse cell line is infected with a helper adenovirus that can
produce virus coat protein but fail to multiplicate.
6. The rDNA gets packed into the protein coat to form infective virus
particles.
7. A sample of the recombinant virus particles are injected into muscle
of mice with defective dystrophin gene.
8. If muscular dystrophy in mice is cured, the viral particles are injected
into human muscle cells to cure muscular dystrophy.
9. The muscle cells intake the rDNA and start to produce dystrophin. As
a result the disease is corrected.
Antisense Gene Therapy:
The treatment of genetic diseases by introducing a remedial gene that prevents
the expression of the specific defective gene is called antisense gene therapy or
antisense therapy.
The defective gene in the patient’s cells produces mRNA by transcription.
The mRNA gets translated into a defective protein. Accumulation of this protein
results in a genetic disease.
A single stranded oligonucleotide complementary to a mRNA binds with the
mRNA and forms an RNA-DNA hybrid. The DNA strand blocks the translation
initiation signals in the mRNA so the defective protein has not been produced.
The oligonucleotide strand blocking the translation of mRNA of defective gene is
called antisense DNA.
In the treatment a duplex DNA similar to the defective gene is introduced into
the cell. The DNA transcribes mRNA which in turn binds with mRNA produced
from the defective gene to form a RNA-RNA hybrid. As a result, defective protein
is reduced in the cell. As mRNA of inserted DNA inhibits the functioning of mRNA
of defective gene it is called antisense RNA.
Genetic Counselling
Advice being given to couples to have healthy babies and to abort fetus that has
genetic disorder after prenatal screening is called genetic counseling. The
physician who is counseling about genetic diseases of foetus is known as genetic
counselor. This method is helpful to breed healthy children and to reduce people
with genetic defects in future.
There are over 1300 genetic defects in human populations. Some of the defects
are due to dominant mutation. Some are due to recessive mutation, and some
are due to linked mutation.
Persons suffering genetic defects due to recessive mutation, die due to severity
of the disease.
Diseases due to dominant mutations have not been expressed in heterozygous
condition. Adults with such diseases seem to be normal, but carriers. When a
heterozygous male marries a homozygous recessive female or vise versa, the
children might be either carriers or sufferers. The sufferers will possess severe
illness due to the genetic defect.
Genetic counselors, carry out prenatal genetic screening and advise the couple
not to have the baby, if any genetic defect prevailing in the foetus.
Genetic counselors carry out prenatal genetic screening and advise the couple
not to have the baby, if any genetic defect is prevailing in the foetus.
1. Sickle cell anemia
2. Huntigtons disease
3. Familial hypercholesterolemia
4. Severe combined immunodeficiency (SCID)
5. Phenylketonuria
6. Emphysema
7. Cystic fibrosis
8. Duchenne muscular dystrophy
9. Hyperammoneuria
10. Thalassemia
11. Tay-Sach’s disease
12. XYY syndrome.
Chorionic villi sampling (SVS), Amniocentesis, fetoscopy, maternal blood
sampling and ultrasound are being used for diagnosis of anatomical
malformations and chromosomal abnormalities in the foetus. Then DNA of
amniotic cells or chorionic villi cells is isolated, amplified using PCR and hybridized
with known probes to determine genetic defects.
If the DNA hybridization is positive, it indicates the genetic defect in the
foetus. Then the genetic counselors advise and educate the couple about the risks
of the genetic defect, current treatments available and about the chance for
inheritance in future generations. They are advising the parents not to give birth
to such babies. The couple aborts the foetus.
Genetic counselling helps to-
1. Avoid giving birth to babies with genetic defect.
2. Prevent risks of newborn babies.
3. Have healthy babies.
SOUTHERN BLOTTING TECHNIQUE
E.Southern in 1975 devised this technique to separate DNA fragments of an
organism based on their size. This method is also called Southern transfer.
It includes the following steps:
The source DNA is cut with a restriction enzyme to generate many small DNA
fragments which differ in their size.
1. The restriction digest is electrophoresed in agarose gel to separate
the DNA fragments depending on their size.
2. The electrophoresed agarose gel is immersed in a depurination
buffer (0.25 M Hcl) for 15 minutes to remove dye components. The
gel is then washed with water.
3. It is then immersed in a denaturation buffer containing 0.5m NaOH
and 1.0M Nacl, for 15 minutes. This alkaline buffer denatures the
duplex DNA into single strands. The gel is washed with water.
4. The gel is then kept immersed in a neutralization buffer to lower the
pH upto 8.
5. A stack of filter paper is placed in a baking dish and SSC* solution is
poured into the dish upto 2/3 height of the filter stack.
6. The pre-treated agarose gel is transferred on the filter stack. A
nitrocellulose filter or nylon membrane is placed on the what man
filters. At the top a glass plate is placed. A proper weight is given to
the glass plate by placing a weight. The experimental set up is kept
as such for 4-8 hours. When the SSC moves to the top layers of
filters, it picks the denatured DNAs in the gel to the nitrocellulose
filter. The DNAs get bound with the nitrocellulose filter.
7. Connect two DNA cross breed (or) Artificial-DNA sequence (Known
sequence (or) complicates gene sequence.
8. The nitrocellulose filter is baked in between filter papers at 80oc for
2 hours.
9. It is then placed in a heat resistant bag containing a pre-
hybridization solution.
10. The pre-hybridization solution is taken out from the bag. The bag is
refilled with a hybridization solution and radioactive labeled DNA
probe or RNA probe. After sealing, the bag is kept at 420C for 4-8
hours for hybridization. The probe hybridizes the complementary
DNA strand on the filter to form a DNA-DNA hybrid or DNA-RNA
hybrid.
11. The nitrocellulose filter is taken out and washed with a wash
solution to remove unbound probes.
12. Autoradiogram is taken by exposing the nitrocellulose filter to an x-
ray film.
Northern Blotting
Northern blotting method was devised by Alwine and his colleagues in 1979 to
separate selective mRNAs from a sample. It is a slight variant of southern blotting.
mRNA is isolated from a cell line at a particular stage and electrophoresed in
agarose gel electrophoresis. The electrophoresed gel is immersed in a
depurination buffer for 10 minutes and then washed with water. The mRNAs in
the garose gel are transferred to an aminobenzyloxymethyl filter paper by usual
blotting method. The mRNA blotted filter paper is baked at 800 C between
Whatmann no.1 filter papers. The blotted filter is treated with a pre-hybridization
solution and then placed in a heat resistant bag. A particular DNA or RNA probe
and hybridization probe are filled in the bag and the bag is sealed. The bag is kept
at 420C for 4-8 hours to establish hybridization. The filter is then washed with a
wash solution to remove unbound probes. An autoradiogram is taken from the
filter to know the exact position of the filter having hybrid nucleic acids.
Western Blotting
Northern blotting method was devised by Alwine and his colleagues in 1979
to separate selective mRNAs from a sample. It is a slight variant of southern
blotting. mRNA is isolated from a cell line at a particular stage and
electrophoresed in agarose gel electrophoresis. The electrophoresed gel is
immersed in a depurination buffer for 10 minutes and then washed with water.
The mRNAs in the garose gel are transferred to an aminobenzyloxymethyl filter
paper by usual blotting method. The mRNA blotted filter paper is baked at 800C
between Whatmann no.1 dilter papers. The blotted filter is treated with a pre-
hybridization solution and then placed in a heat resistant bag. A particular DNA or
RNA probe and hybridization probe are filled in the bag and the bag is sealed. The
bag is kept at 420 for 4-8 hours to establish hybridization. The filter is then washed
with a wash solution to remove unbound probes. An autoradiogram is taken from
the filter to know the exact position of the filter having bybrid nucleic acids.
Western Blotting
Western blotting method was devised by Tow bin in 1979 to find out the
protein encoded by the cloned gene in conformed cells. It is an immunodetection
method working in principle of antigen-antibody reaction. It does not need
radiolabelled nucleic acid probes. The simplified steps of western blotting are
outlined here under:
1. Proteins are extracted from a cell (transformed cell).
2. The protein isolate is electrophoresed in polyacrylamide gel to separate
various proteins based on their size. Sodium dodecyl sulphate (SDS) is
used as solvent for electrophoresis.
3. The electrophoresed gel is immersed in a transfel buffer (TGM buffer) for
30 minutes at 40c.
4. Nitrocellulose filter, whatmann filters and coars filters are soaked well
with TGM buffer.
5. A stack of coarse filters is kept in between two whatmann filters. It is
placed on the cathode plate of a semidry Western blot apparatus. The
elelctrophoresed gel is transferred onto the filter stack. A nitrocellulose
filter is placed on the gel. Another stack of filter papers is placed on the
nitrocellulose filter. The anode plate is kept on the filter stack.
6. A 500 mA current is supplied to the apparatus for 1 hour. During this time
proteins are transferred from the ployacrylamide gel to the nitrocellulose
filter.
7. The nitrocellulose filter is taken and kept immersed in a backing solution
for 30 minutes at 370c. The blocking solution contains bovine serum
albumin (BSA) or milk powder. This protein binds with unoccupied sites on
the filter for blocking the binding of antibodies with the filter.
8. Antibody against the target protein to be detected is prepared from rabbit
and labelled with radioactive I123.
9. After blocking, the nitrocellulose filter is placed in the solution containing
radialobelled antibodies for 12 hours.
10. The nitrocellulose filter is washed with a wash solution (Tris-buffered
saline+Tween 20) to remove unbound antibodies.
11. The filter is exposed to an x-ray film for 4 hours to take an autoradiogram.
Dots in the radiogram indicate the presence of target protein in the
protein isolate.
USES:
1. It is an extremely sensitive analytical method that can detect target
protein even if it is present as low as 5 mg in a crude sample.
2. The quantity of the gene product (protein) can also be detected.
3. Species specific differences in proteins can be detected.
UNIT - V
CONCEPTS OF TRANSGENIC ANIMAL TECHNOLOGY
Genetically manipulated animals having introduced gene in their genetic make up are called
transgenic animals.
They are also known as transgenics.
Transgenic mice, sheep, cattle, goats, rabbits, poultry, fishes, mosquitoes, etc., have been
developed by using genetic engineering methods.
The desired genes are transferred to eggs, zygotes, embryonic stem cells or blastoderm cells.
Usually microinjection retroviral infection and transfection are used to transfer genes to the
cells.
The introduced gene expresses its character in the transgenics. It is inherited for many
generations.
Transgenic animals produce some noval proteins of therapeutic value, more milk and fast
growth.
STRATEGIES FOR THE PRODUCTION OF TRANSGENIC ANIMALS AND THEIR IMPORTANCE IN
BIOTECHNOLOGY
RETROVIRAL METHOD
Introduction of foreign DNA into eggs with the help of a retrovirus vector is called retroviral
method.
Small foreign genes, less than 8kbp. Can be transferred to recipient cells through retrovirus
vector.
Murine leukaemia virus (MuLV) is the most common retrovirus being used to introduce genes
into animal cells. The retrovirus transgenic methodology is given below.
A mouse cell line is infected with murine leukaemia virus (MuLV) . RNA of murine leukaemia
virus (MuLV)produces double standard DNA by reverse transcription.
This proviral DNA is isolated from the cell line.
The foreign gene is linked with a neomycin resistance gene using DNA ligase.
The foreign gene (DNA) is inserted into the proviral DNA using a proper restriction enzyme and
DNA ligase. The resulting hybrid DNA is called rDNA.
The rDNA is introduced with a helper murine leukaemia virus (MuLV) that can produce viral
capsid but cannot replicate or with a mutant murine leukaemia virus (MuLV) which has no signal
for invivo packaging.
RNA of the helper virus produces viral capsid. The rDNA produces recombinant viral RNA. The
latter gets packaged in the viral capsid to form infective recombinant murine leukaemia virus
(MuLV) particles.recombinant murine leukaemia virus (MuLV)
Egs of mouse are collected and fertilized invitro to form zygotes
On reaching 8-celled stage, the mouse embryos are infected with the murine leukaemia virus
(MuLV) . inside the infected cell the recombinant RNA is reverse transcribed into double
stranded DNA. The DNA integrates with the cell DNA and hence recombinant cells are formed.
The mouse embryos are implanted into surrogate mothers and the mice and fed well.
The surrogate mother mice produce transgenic mouse siblings with the foreign gene insert.
MICROINJECTION METHOD
In microinjection method foreign genes are injected into a fertilized egg by a micropipette. The
important steps of this method are outlined below.
A female mouse is superovulated by injecting pregnant mare’s serum and human chorionic
gonadotropin. The mouse produces about 35 eggs.
This female mouse is mated with a male
Fertilized eggs are taken from the oviduct and kept in a balanced salt solution.
The eggs are observed under the microscope to pick up eggs having male and female pronuclei,
ie. Just before the fusion of male and female nuclei. The male pronucleus is larger than the
female one.
The foreign gene is microinjected into the male pronucleus of the eggs with the help of a
micropipette.
About 25-40 microinjected eggs are implanted in the uterus of a foster mother and the female is
then mated with a vasectomized male. This mating brings up a pseudopregnant condition for
the further development of the eggs in the uterus.
The foster mother will give birth to transgenic pups.
Transgenic pups can be screened by southern blotting. DNA isolated from small piece of tail is
hybridized with known probe to detect the transgenic mice.
STRATEGIES FOR THE PRODUCTION OF TRANSGENIC ANIMALS AND THEIR IMPORTANCE IN
BIOTECHNOLOGY
RETROVIRAL METHOD
Introduction of foreign DNA into eggs with the help of a retrovirus vector is called retroviral
method.
Small foreign genes, less than 8kbp. Can be transferred to recipient cells through retrovirus
vector.
Murine leukaemia virus (MuLV) is the most common retrovirus being used to introduce genes
into animal cells. The retrovirus transgenic methodology is given below.
A mouse cell line is infected with murine leukaemia virus (MuLV) . RNA of murine leukaemia
virus (MuLV)produces double standard DNA by reverse transcription.
This proviral DNA is isolated from the cell line.
The foreign gene is linked with a neomycin resistance gene using DNA ligase.
The foreign gene (DNA) is inserted into the proviral DNA using a proper restriction enzyme and
DNA ligase. The resulting hybrid DNA is called rDNA.
The rDNA is introduced with a helper murine leukaemia virus (MuLV) that can produce viral
capsid but cannot replicate or with a mutant murine leukaemia virus (MuLV) which has no signal
for invivo packaging.
RNA of the helper virus produces viral capsid. The rDNA produces recombinant viral RNA. The
latter gets packaged in the viral capsid to form infective recombinant murine leukaemia virus
(MuLV) particles.recombinant murine leukaemia virus (MuLV)
Egs of mouse are collected and fertilized invitro to form zygotes
On reaching 8-celled stage, the mouse embryos are infected with the murine leukaemia virus
(MuLV) . inside the infected cell the recombinant RNA is reverse transcribed into double
stranded DNA. The DNA integrates with the cell DNA and hence recombinant cells are formed.
The mouse embryos are implanted into surrogate mothers and the mice and fed well.
The surrogate mother mice produce transgenic mouse siblings with the foreign gene insert.
MICROINJECTION METHOD
In microinjection method foreign genes are injected into a fertilized egg by a micropipette. The
important steps of this method are outlined below.
A female mouse is superovulated by injecting pregnant mare’s serum and human chorionic
gonadotropin. The mouse produces about 35 eggs.
This female mouse is mated with a male
Fertilized eggs are taken from the oviduct and kept in a balanced salt solution.
The eggs are observed under the microscope to pick up eggs having male and female pronuclei,
ie. Just before the fusion of male and female nuclei. The male pronucleus is larger than the
female one.
The foreign gene is microinjected into the male pronucleus of the eggs with the help of a
micropipette.
About 25-40 microinjected eggs are implanted in the uterus of a foster mother and the female is
then mated with a vasectomized male. This mating brings up a pseudopregnant condition for
the further development of the eggs in the uterus.
The foster mother will give birth to transgenic pups.
Transgenic pups can be screened by southern blotting. DNA isolated from small piece of tail is
hybridized with known probe to detect the transgenic mice.
TRANSGENIC SHEEP:
Genetically manipulated sheep having introduced gene in their genetic makeup
is called transgenic sheep.
Microinjection method has been used to create transgentic sheep with novel
gene construct.
Mammary glands of the transgenic sheep serve as bioreactors and produce
pharmaceutically valuable proteins. Eg.α-1 antitrypsin (ATT). Factor IX, etc.
Production of human proteins from the milk of transgenic animals is called animal
pharming.
The α-1 antitrypsin gene is fused with a β-lactoglobin promoter and is
microinjected into male pronucleus of fertilized egg.
The sheep produces transgenic lambs. The transgenic lambs, after reaching
maturity, give 1-35g of α-1 antitrypsin in a litre of milk.
Emphysema, a lung disease in man can be treated by injecting α-1 antitrypsin.
Transgenic sheep with gene for factor IX is produces factor IX in the milk .
Factor IX is isolate from the milk, purified and used to treat haemophilia in man .
For having high rate of wool production, sheeps have been genetically
manipulated to have growth hormone gene or keratin gene or cystine gene. The
transgenic sheep produce more high quality wool.
TRANSGENIC PIGS
Gene for Factor VIII was introduced into zygote of pig by microinjection and the
zygote was implanted in the uterus of a sow. The sow produce transgenic pigs
that could produce Factor VIII in the milk. The Factor VIII has been isolated and
purified from the milk and used to treat haemophilia.
The transgenic pigs with human β-globulin gene produce human haemoglobin
in their blood. Attempts have been taken to use haemoglobin for transfusion.
XENOTRANSPLANTATION:
Transplantation of animal organs in human system is called
xenotransplantation. Organs of pigs were tried for xenotransplantation as they
are in the same size as the human organs. But the human body produced
antibodies against the pig organs and rejected the transplants. This is called
hyperacute rejection.
A team of researchers at Imutran Company (USA) microinjected certain genes
from human immune system into male pronucleus of pig zygote. They introduced
the zygote into a sow to produce transgenic pig. The transgenic pigs had human
proteins so that human body may accept their organs. The transgenic pig was
named Astrid. Attempts have been taken to transplant heart, kidney, liver, lungs
and pancreas of transgenic pigs in human beings.
TRANSGENIC FISHES
Genetically manipulated fishes having introduced gene in their genetic makeup
are called transgenic fishes.
Desired foreign genes are introduced into four celled fish embryos by
microinjection.
The embryos are growth into transgenic fishes in temperature controlled
tanks.
Transgenic fishes such as carps, catfishes, salmons and Tilapia have been
developed by using this method.
Transgenic gold fish was made by introducing human growth hormone gene
into immature embryos. They grew faster than the normal gold fishes.
A gene for antifreeze protein (AFP) of a winter flounder was transferred to
atlantic salmon. The transgenic salmons can tolerate low temperature.
In fishes fertilization and development are external. The rDNA is introduced
into the zygote by microinjection. The recombinant zygote is grown into a
transgenic fingerling. The following transgenic methodology carps, catfishes,
salmons, Tilapia, etc.
1. Freshly laid fish eggs are collected from water.
2. The foreign gene is microinjected into the cytoplasm of the eggs or 4-celled
embryos.
3. The zygotes or embryos are cultured separately in a temperature controlled
tank.
4. The embryos develop into transgenic fishes.
This transgenic methodology is relatively simple. There may be 35%
mortality of engineered zygotes or embryos. Hence 10-70% transgenic
fishes can be produced in this way.
Biosensors
A biosensor is a sensitive analytical tool which converts biological signals
provided by the analyte into electrical signals.
Types of Biosensors
Biosensors are usually classified into the following types:
1. ISFET based biosensors
2. Enzyme electrodes
3. Immobilized cell biosensors
4. Thermal biosensors
ION-SENSITIVE FIELD EFFECT TRANSISTOR (ISFET) BASED BIOSENSORS:
This type of biosensors has semiconducting device called field effect transistor
(FET). The FET consists of silicon crystals. It has high electrical resistance unless
the electric field is modified.
Enzyme Electrodes:
In this type of biosensors, an enzyme is immobilized onto the surface of an
electrode. The enzyme catalyses its specific reaction and thus transfers electrons
from the reactants to the electrode. Current generated on the electrode is
amplified, read out and recorded.
Immobilized Cell Biosensors:
In these biosensors the whole cells are immobilized on the electrodes, they are
also known as microbial biosensors or whole cells biosensors.
To measure ethanol, the yeast Trichosporon brassicae is immobilized on the
electrode. This biosensor can measure low concentration of ethanol, ie. Less
than 22.5 mg dm-3, within 10 minutes.
Bacteria which metabolise amino acids are immobilized in amino acid
biosensors. The yeast Trichosporon cutaneum is immobilized in BOD biosensors.
Bacteria resistant to heavy metals are used in heavy metals biosensors.
Cells of BGA are immobilized in herbicide biosensors.
Thermal Biosensors:
Thermal biosensors are used to detect small changes in temperature in
enzyme catalyzed reactions. Low temperatures between 0.0010C can also be
measured with thermal biosensors. They are constructed by immobilizing a
suitable enzyme on an electrode.
APPLICATIONS OF BIOSENSORS
Biosensors are suitable systems for on-line measurements and continuous
operations. They are small in size, easy to work, quick in action, very sensitive to
measure tiny amount of test molecules and relatively very cheap. So they are put
into many practical uses in medicine, industries and environmental control.
Medicine:
A glucose biosensor coupled with a minipump containing insulin, is used to
detect blood glucose level directly and to deliver the accurate amount of insulin
required by the diabetic. Usually enzyme electrode is used for this purpose.
Biosensors are used to detect mutagenic substance in the body fluid.A few
biosensors are employed to detect toxic substances and heavy metals in blood
metals in blood samples.
Industries:
Biosensors are suitable for on-line measurements of certain compounds in
continuous industrial process. As they are very sensitive and quick in action, they
have been put into many practical uses in industries.
The following are some important uses of biosensors in industries:
A microbial sensor made up of immobilized cells of pseudomonas
fluorescens and an oxygen electrode, is used to detect glucose level
in molasses.
The total amount of assimilable sugars in fermentation media and
broths can be measured with a microbial sensor. This biosensors is
made up of an immobilized layer of Brevibacterium
lactofermentum AJ 1511 and an oxygen electrode.
An acetic acid sensor is made by combining an immobilized layer
Trichosporon brassicae and an oxygen electrode. It detects acetic
acide level in fermentation broths for glutamic acid within 6-10
minutes. Acetic acid level less than 22.5 mg dm-3 can also be
detected sensitively.
The cell number in fermentation broth and various food items can
be measured using fuel cell type biosensors. This biosensor consists
of two electrodes, each of which is made of a platinum anode and
silver peroxide cathode. The electrochemical changes caused by the
microbes in the analyte are used to measure the cell number of
Saccharomyces cerevisiae and Lactobacillus fermentum.
The cell number of Bacillus subtilis in fermentation broth can be
measured with potentiometric biosensors.
A lactate sensor is used to count lactic acid producing bacteria in
fermentation media. The lactate sensor is made up of an
immobilized layer of lactate oxidase and a platinum electrode
coupled with a volumetric device. Leuconostoc, Streptococcus and
Lactobacillus are counted in this way.
An alcohol sensor is made up of an immobilized layer of
Trichosporon brassicae methyl alcohol and ethyl alcohol in
fermentation broth and beverages.
A formic acid sensor is made with immobilized layer of Clostridium
butrycum and a fuel cell type electrode. It is used to measure
formic acid level in fermentation broths.
Freeze dried cells of E.coli are immobilized on a silicon chip using a
cellophane membrane. This sensor analyses glutamic acid in
fermentation media.
A cephalosporin sensor is made by entrapping the bacterium
Citrobacter freundii on a glas electrode using a collagen membrane.
It is used to measure cephalosporin.
A nystatin sensor is made by entrapping the yeast Saccharomyces
cerevisiae on an oxygen electrode using a collagen membrane. This
is used to detect the concentration of nystatin, in fermentation
media.
A vitamin B1 sensor is made by immobilizing Lactobacillus fermenti
(ATCC 9338) on a platinum electrode. It measures vitamin B1 level
in fermentation broths.
A nicotinic acid sensor is assembled by immobiliszing Lactobacillus
arabinosus (ATCC 8014) on an oxygen electrode by using agar gel. It
measures nicotinic acid level in fermentation media.
Isaokarube and his colleagues have made an ISFET based biosensor
using the enzyme ATPase, aminoxidase or putrescine oxidase. It
detects the freshness of fishes and animal fleshes for cooking.
The UK based Cransfield Institute of Technologly has developed a
cholesterol sensor to detect cholesterol level in food items. This
biosensor is made up of an immobilized layer of the enzyme
cholesterol oxidase and an electrode.
1. Environmental Control:
Biosensors have been employed to detect the concentration of
pollutants before going treat polluted waters. As they can be automated,
the quality control of drinking water and the assessment of pollution status
are easy to workout sensitively.
The following sensors are of much use in environmental control:
In Japan, a BOD sensor was made by immobilizing the yeast
Trichosporon cutaneum on an oxygen electrode using a porous
acetylcellulose membrane. It measures BOD of water samples
within 18 minutes. Further, it can be adopted to use contiuously
for 17 days.
An ammonia sensor is made by coupling an immobilized layer of
Nitrosomonas cells on oxygen electrodes. It measures ammonia
level in water samples.
A nitrate sensor is made by coupling an immobilized layer of
Nitrobacter on an oxygen electrode. Nitric oxide (NO) and
nitrogen oxide (NO2) can be detected by this sensor.
A sulfite sensor is made by immobilizing microsomes of Rat liver
cells on an oxygen delctrode by using a porus acetylcellulose
membrane. It is used to detect sulfite and sulfur dioxide pollution
in waters.
A dye-coupled electrode system is made by using a fuel cell
electrode, a redox dye and a porous acetylcellulose membrane. It
is used to detect cell number of E.coli in polluted waters.
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