SHREE SIDDAGANGA COLLEGE OF SHREE SIDDAGANGA COLLEGE OF PHARMACY TUMKUR, KARNATAKAPHARMACY TUMKUR, KARNATAKA
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ContentContentss
HistoryHistory DefinitionDefinition Steps in cloning Steps in cloning o Generating DNA fragmentsGenerating DNA fragments
o Insertion of target DNA in to vectorInsertion of target DNA in to vector
o Introduction of recombinant DNA in to host cellIntroduction of recombinant DNA in to host cell
Applications of cloning
References
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History:History:
1869 - Miescher – isolated DNA from 1869 - Miescher – isolated DNA from WBC.WBC.
1944 - Avery - DNA is primary genetic 1944 - Avery - DNA is primary genetic material.material.
1953 - Watson Crick - DNA double helix 1953 - Watson Crick - DNA double helix structure.structure.
1962 - Arber - evidence of DNA 1962 - Arber - evidence of DNA restriction nuclease.restriction nuclease.
1966 - Nirenberg Ochoa and Khorana - 1966 - Nirenberg Ochoa and Khorana - elucidation of genetic code. elucidation of genetic code.
1967 - Gellert - DNA ligase enzyme.1967 - Gellert - DNA ligase enzyme. 1972 - 73 - DNA Cloning techniques. 1972 - 73 - DNA Cloning techniques. SSCP TUMKURSSCP TUMKUR
Definition:Definition:
– Cutting a piece of DNA from one Cutting a piece of DNA from one organism and inserting it into a organism and inserting it into a vector where it can be replicated by vector where it can be replicated by a host organism. (Sometimes called a host organism. (Sometimes called subcloning, because only part of the subcloning, because only part of the organism’s DNA is being cloned.)organism’s DNA is being cloned.)
– Using nuclear DNA from one Using nuclear DNA from one organism to create a second organism to create a second organism with the same nuclear DNAorganism with the same nuclear DNA
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Main steps in Main steps in cloning:cloning:
•Generating DNA fragmentsGenerating DNA fragments
•Insertion of target DNA in to vectorInsertion of target DNA in to vector
•Introduction of recombinant DNA in Introduction of recombinant DNA in to host cellto host cell
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Generating DNA fragments Generating DNA fragments ::
Mechanical shearing:Mechanical shearing:• High speed mixing in blender or High speed mixing in blender or
sonicator.sonicator.• Random fragment of source DNA.Random fragment of source DNA.• Does not produce 5’phasphate and Does not produce 5’phasphate and
3’OH ends.3’OH ends.• Requires repair the ends (S1nuclease Requires repair the ends (S1nuclease
or DNA polymerase).or DNA polymerase).• Not reproducible in nature.Not reproducible in nature.
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Restriction Endonuclease Digestion:Restriction Endonuclease Digestion:Restriction endonuclease are the Restriction endonuclease are the
enzymes isolated from bacteria that cut enzymes isolated from bacteria that cut DNA at specific recognition nucleotide DNA at specific recognition nucleotide sequences known as restriction sites.sequences known as restriction sites.
• More than 200 restriction enzymes.More than 200 restriction enzymes.• Size of DNA fragments depends upon Size of DNA fragments depends upon
restriction sites present in DNA.restriction sites present in DNA.• It produces either sticky end or blunt It produces either sticky end or blunt
ends.ends.• Generally same enzyme is used for the Generally same enzyme is used for the
vector and the DNA of interest.vector and the DNA of interest.• Reproducibility of fragment is possible. Reproducibility of fragment is possible.
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- Restriction enzymes can generate both sticky ends and blunt ends after they cut the recognition sequence
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Reverse Transcriptase Method:Reverse Transcriptase Method:• Gene organisation of eukaryotes and Gene organisation of eukaryotes and
prokaryotes is different.prokaryotes is different.
• Bacteria or yeast do not have necessary Bacteria or yeast do not have necessary splicing mechanism for removal of splicing mechanism for removal of introns.introns.
• Mature mRNA molecules from animal cell Mature mRNA molecules from animal cell can be directly transcribed in to DNA can be directly transcribed in to DNA using an enzyme reverse transcriptase.using an enzyme reverse transcriptase.
• cDNA produced for particular protein can cDNA produced for particular protein can be joined to vector and cloned in to host be joined to vector and cloned in to host cell.cell.
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Hybridization Method:Hybridization Method:
• Principle: mRNA forms a complex with Principle: mRNA forms a complex with complementary DNA segments from complementary DNA segments from which it has been transcribed.which it has been transcribed.
• Total DNA from donor organism is Total DNA from donor organism is isolated & dsDNA is converted to ssDNA isolated & dsDNA is converted to ssDNA by denaturation.by denaturation.
• Mixed with mRAN trancribed by the gene Mixed with mRAN trancribed by the gene this forms DNA-RNA complex (Hybrid).this forms DNA-RNA complex (Hybrid).SSCP TUMKURSSCP TUMKUR
Cont…Cont…
• DNA-RAN complex is isolated & DNA DNA-RAN complex is isolated & DNA is separated from RNA.is separated from RNA.
• ssDNA obtained can be converted to ssDNA obtained can be converted to dsDNA by DNA polymerase I.dsDNA by DNA polymerase I.
• Suitable for isolating genes which Suitable for isolating genes which exist in multiple copies. exist in multiple copies.
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Chemical Synthesis:Chemical Synthesis:• The technique is termed as reverse The technique is termed as reverse
genetics.genetics.
• Possible only when amino acid sequence of Possible only when amino acid sequence of protein of interest is known.protein of interest is known.
• For which sequence of nucleotides in For which sequence of nucleotides in mRNA & subsequently in DNA can be mRNA & subsequently in DNA can be determined.determined.
• This method of generating DNA fragments This method of generating DNA fragments for cloning will be batter for creating novel for cloning will be batter for creating novel proteins.proteins. SSCP TUMKURSSCP TUMKUR
IDENTIFICATION OF CLONE
SOUTHERN
BLOT
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Insertion of Target DNA into Insertion of Target DNA into Vector:Vector:
Vectors: are Vectors: are carrier DNAscarrier DNAs into which into which foreign DNAsforeign DNAs are spliced to make a are spliced to make a recombinant DNA.recombinant DNA.
Types of vectors:Types of vectors:• Plasmids
• Bacteriophages
• Cosmids
• BACs & YACsSSCP TUMKURSSCP TUMKUR
VECTOR INSERT SIZE VECTOR INSERT SIZE (kbp)(kbp)
• Plasmid ~ 15.0 kbpPlasmid ~ 15.0 kbp • λ Phage ~ 50.0 kbpλ Phage ~ 50.0 kbp • Cosmid > 50.0 <100.0 Cosmid > 50.0 <100.0
kbpkbp • Bacterial artificial ~ 300.0 kbp Bacterial artificial ~ 300.0 kbp
Chromosome (BAC) Chromosome (BAC) • Yeast artificial ~ 2000 kbp Yeast artificial ~ 2000 kbp
Chromosome (YAC)Chromosome (YAC)
Selection of Vector:
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PlasmidsPlasmids Plasmids are double-stranded, circular, Plasmids are double-stranded, circular,
self-replicating, extra-chromosomal self-replicating, extra-chromosomal DNA molecules.DNA molecules.
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• Plasmids are circular pieces of DNA Plasmids are circular pieces of DNA found naturally in bacteria.found naturally in bacteria.
• Plasmids can carry antibiotic Plasmids can carry antibiotic resistance genes, genes for resistance genes, genes for receptors, toxins or other proteins.receptors, toxins or other proteins.
• Plasmids Plasmids replicate separately from separately from the genome of the organism.the genome of the organism.
• Plasmids can be engineered to be Plasmids can be engineered to be useful useful cloning vectors..
Cont…
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Isolation of bacterial plasmidsIsolation of bacterial plasmidso Treat the prokaryotic cells with detergent.Treat the prokaryotic cells with detergent.
o Treat the lysate with potassium acetate/ Treat the lysate with potassium acetate/ acetic acid solution and centrifuge.acetic acid solution and centrifuge.
o Treat the lysate with RNAase consequently.Treat the lysate with RNAase consequently.
o Mix the lysate with phenol. Phenol & water Mix the lysate with phenol. Phenol & water is separated in two layers . The water is separated in two layers . The water based layer contains plasmid DNA.based layer contains plasmid DNA.
o Precipitate water based layer with alcohol Precipitate water based layer with alcohol and collect the plasmid DNA.and collect the plasmid DNA.
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Ex. Of plasmidsEx. Of plasmids
• pBR 322- derived from E.coli prepared pBR 322- derived from E.coli prepared by by BBolivar and olivar and RRodriguez.odriguez.
• pAT 153 and pXf 3 are two derivatives pAT 153 and pXf 3 are two derivatives of pBR 322 having smaller size & of pBR 322 having smaller size & higher copy.higher copy.
• pUC derived from E.coli initial pUC derived from E.coli initial prepared in prepared in UUniversity of niversity of CCalifornia.alifornia.
Cont…..
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Advantages of plasmids:Advantages of plasmids:– Small, easy to handleSmall, easy to handle– Useful for cloning small DNA Useful for cloning small DNA
fragments fragments
(< 15kbp)(< 15kbp) Disadvantages of plasmids:Disadvantages of plasmids:
– Less useful for cloning large DNA Less useful for cloning large DNA fragments fragments
(> 15kbp)(> 15kbp)
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BacteriophageBacteriophagess
• Phases has linear DNA, single break create two fragments.
• Foreign DNA can be inserted between them & two fragments can be joined.
• Bacteriophages act as hypodermic syringe like and insert DNA in to the host and replicates.
• Lytic Cycle: rapid infection resulting in lysis of cell and release of multiple bacteriophages.
•Lysogenic phages: For bacteriophage λ, DNA integrates in to bacterium genome.
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BACTERIOPHAGES
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CosmidsCosmids• Cosmids posses properties of both plasmids Cosmids posses properties of both plasmids
and bacteriophages.and bacteriophages.
• Contain a cos site of Contain a cos site of λλ phage (essential for phage (essential for packaging of nucleic acid into protein coat)packaging of nucleic acid into protein coat)
• plus essential features of plasmids ( plasmid plus essential features of plasmids ( plasmid origin of replication, a gene for resistance)origin of replication, a gene for resistance)
• Cosmids can be constructed by adding a fragment of phage λ DNA including cos site to plasmid.
• A foregin DNA can be inserted into cosmid DNA
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COSMIDS
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BACs: Bacterial Artificial BACs: Bacterial Artificial ChromosomesChromosomes
The construction BAC F
plasmid, which is large than other plasmid used as cloning vector.
BACs can accept DNA inserts of around 300kb
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YACs:Yeast Artificial ChromosomesYACs:Yeast Artificial Chromosomes• YAC are the most
sophisticated yeast vectors.
• They have centromeric & telomeric region of chromosome.
• very large piece of DNA can be cloned (particular human DNA) ~ 2000 kbp.
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LigationLigation• Ligation is the process of joining two is the process of joining two
pieces of DNA from different sources pieces of DNA from different sources together through the together through the formation of a covalent bond. .
• DNA ligase is the enzyme used to DNA ligase is the enzyme used to catalyze this reaction.catalyze this reaction.
• These enzymes are originally isolated These enzymes are originally isolated from from virusesviruses, also occur in E.coli & , also occur in E.coli & eukaryotic cell.eukaryotic cell. SSCP TUMKURSSCP TUMKUR
DNA Ligases
Type Energy source Used for:E. coli ligase
NAD (turned into AMP and NMN)
Sticky ends
T7 ligase ATP (turned into AMP and PPi)
Sticky ends
T4 ligase ATP (turned into AMP and PPi)
Both sticky and blunt ends
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• The most important precaution prior to setting up a ligation reaction is to ensure that the cut vector is prevented from recircularization.
• This is achieved by digesting the plasmid by the same restriction enzyme(s).
• Treatment with alkaline phosphatase which removes the terminal phosphate groups from the cut ends of the plasmid. This ensures that the cut plasmid does not recircularize.
PRECAUTION
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COHESIVE
LIGATION
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BLUNT END LIGATIONBLUNT END LIGATION
Homopolymer tailingHomopolymer tailing
Use of Linkers MoleculeUse of Linkers Molecule
Use of Adaptors MoleculeUse of Adaptors Molecule
The blunt end DNA can be joined by :
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Homopolymer Homopolymer tailingtailing
• The complementary DNA strands can be The complementary DNA strands can be joined together by annealing.joined together by annealing.
• Addition of oligo (dA) to 3’ ends of some Addition of oligo (dA) to 3’ ends of some DNA molecule and addition of oligo (dT) DNA molecule and addition of oligo (dT) to 3’ ends of other molecule.to 3’ ends of other molecule.
• The homopolymer extension can be The homopolymer extension can be synthesized by using terminal synthesized by using terminal deoxynucle- otidyltrasferase (calf deoxynucle- otidyltrasferase (calf thymus).thymus).
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5’ 5’
Insert Vector
3’ 3’
3’ 3’
5’5’
dATP Terminal deoxynucleotidyltransferas
edTTP
5’5’AAA 3’
3’AAA
3’ TTTTTT 3’
LIGATION
5’ 5’
Homopolymer tailingHomopolymer tailing
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Use of Linkers MoleculeUse of Linkers Molecule• Linkers are chemically synthesized, short, Linkers are chemically synthesized, short,
double strand DNA molecules.double strand DNA molecules.
• possess restriction enzyme cleavage sites.possess restriction enzyme cleavage sites.
• they can be ligated to blunt ends of any they can be ligated to blunt ends of any DNA.DNA.
• Cut with specific restriction enzymes to Cut with specific restriction enzymes to produce sticky end DNA fragments produce sticky end DNA fragments
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Blunt- ended insert5’
5’3’
3’Linker (e.g. Bam HI)
5’ P GGGATCCC OH 3’3’ OH CCCTAGGG P 5’
Ligate to linker
GGGATCCCGGGATCCC
GGGATCCCGGGATCCC
CCCTAGGGCCCTAGGG
CCCTAGGGCCCTAGGG
Cut with Bam HI
GG
GG
GATCCC
CCCTAG
Ligate to vector cut with Bam HISSCP TUMKURSSCP TUMKUR
Use of Adaptors MoleculeUse of Adaptors Molecule
• Adaptors are chemically synthesized, Adaptors are chemically synthesized, short, double-stranded DNA molecules. short, double-stranded DNA molecules.
• Adaptor contains preformed sticky or Adaptor contains preformed sticky or cohesive ends.cohesive ends.
• They are useful to be ligated to DNA They are useful to be ligated to DNA fragment with blunt ends.fragment with blunt ends.
• The DNA fragments held to adaptors The DNA fragments held to adaptors are finally ligated to vector DNA are finally ligated to vector DNA molecules. molecules. SSCP TUMKURSSCP TUMKUR
5’
5’
3’
3’GGGATCCCGGGATCCC
CCCTAGGG
CCCTAGGGGGGATCCCGGGATCCC
DNA Fragment Adaptors
DNA ligase+ATP
+
GGGATCCCGGGATCCCCCCTAGG
G
CCCTAGGGGGGATCCCGGGATCCC
Ligate to vectorSSCP TUMKURSSCP TUMKUR
Introduction of recombinant DNA in to hostIntroduction of recombinant DNA in to host cellcell
• After creating new vector construct it After creating new vector construct it needs to insert into a host cell so that it needs to insert into a host cell so that it can be replicate.can be replicate.
• Hosts are the living systems or cells in Hosts are the living systems or cells in which vector can be propagated which vector can be propagated
• The process of introducing the foreign The process of introducing the foreign DNA into the host cell is called DNA into the host cell is called transformationtransformation..
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Cont…Cont…
• Host cell may be prokaryotic (bacteria) Host cell may be prokaryotic (bacteria) or eukaryotic (fungi, animal, plant).or eukaryotic (fungi, animal, plant).
• Micro organisms are preferred as host Micro organisms are preferred as host cells, since they multiply faster.cells, since they multiply faster.
PROKARYOTIC HOST (Bacteria):PROKARYOTIC HOST (Bacteria):• Not every bacterial cell is able to take Not every bacterial cell is able to take
up plasmid DNA.up plasmid DNA.
• Bacterial cells that can take up DNA Bacterial cells that can take up DNA from the environment are said to be from the environment are said to be competentcompetent..
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Escherichia Escherichia coli:coli:
• E.coli was the first organism used in E.coli was the first organism used in the DNA technology experiments.the DNA technology experiments.
• Is common Gram – ve bacterium of Is common Gram – ve bacterium of human and animal intestine.human and animal intestine.
• Under suitable environment E.coli Under suitable environment E.coli multiply two times in every 20 min.multiply two times in every 20 min.
Limitations of E.coli:Limitations of E.coli:- Formation of endotoxins that are toxic.Formation of endotoxins that are toxic.
- Cannot perform post- translational Cannot perform post- translational modificationmodification
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Eukaryotic Eukaryotic hosts:hosts:
• Eukaryotic organisms are preferred to Eukaryotic organisms are preferred to produce human proteins.produce human proteins.
• Most commonly used organism is yeast, Most commonly used organism is yeast, Saccharomyces. Saccharomyces.
• It is non pathogenic organism rutinely used in It is non pathogenic organism rutinely used in baking industry.baking industry.
Mammalian cells:Mammalian cells:- Certain complex protein produced by Certain complex protein produced by
mammalian cells Eg. Tissue plasminogen mammalian cells Eg. Tissue plasminogen activator.activator.
- mammalian cell possess post translation mammalian cell possess post translation modifications.modifications.
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Some Examples of Host cellsSome Examples of Host cells
Group ExamplesGroup Examples
Prokaryotic Prokaryotic Bacteria E.coliBacteria E.coli
Bacillus SubtilisBacillus Subtilis
Streptomyces spStreptomyces sp
EukaryoticEukaryoticFungi Saccharomyces cervisiaeFungi Saccharomyces cervisiae
Aspergillus nidulansAspergillus nidulans
Animals Insect cellsAnimals Insect cells
mammalian cellsmammalian cells
Whole organismsWhole organisms
Plants ProtoplastsPlants Protoplasts
Intact cellIntact cellSSCP TUMKURSSCP TUMKUR
Method of gene Method of gene transfertransfer
Introduction of foreign DNA in to host Introduction of foreign DNA in to host cell is important in gene cloning, cell is important in gene cloning, commonaly employed methods are:commonaly employed methods are:
oTransformation Transformation
oLipofectionLipofection
oTransduction Transduction
oElectroporationElectroporation
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TransformionatTransformionat
• Is a method of introducing foreign Is a method of introducing foreign DNA in to bacterial cells (E.coli)DNA in to bacterial cells (E.coli)
• Carried out in ice-cold CaClCarried out in ice-cold CaCl2 2 (0-5c) & (0-5c) & subsequent heat shock (37- 45c for subsequent heat shock (37- 45c for about 90 sec.)about 90 sec.)
• Some times calcium phosphate or Some times calcium phosphate or diethyl aminoethyl dextran (DEAE-diethyl aminoethyl dextran (DEAE-dextran) is replaced to CaCldextran) is replaced to CaCl2 .2 .
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LipofectionLipofection The liposome mediated gene transfer, is The liposome mediated gene transfer, is
referred as lipofection.referred as lipofection.
• It is vary efficient technique & is used for - It is vary efficient technique & is used for - bacteria, animal & plant host cell.bacteria, animal & plant host cell.
• On treatment of DNA fragment with liposome, On treatment of DNA fragment with liposome, the DNA get encapsulated inside liposome.the DNA get encapsulated inside liposome.
• Liposome can adhere to cell membrane & Liposome can adhere to cell membrane & transfer DNA fragments.transfer DNA fragments.
• DNA enters the cell & then to the nucleus.DNA enters the cell & then to the nucleus.
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TransductionTransduction
The foreign DNA can be packed The foreign DNA can be packed inside animal viruses.inside animal viruses.
These viruses can naturally infect the These viruses can naturally infect the cells & introduce the DNA into host cells & introduce the DNA into host cell.cell.
The transfer of DNA by this approach The transfer of DNA by this approach is referred as transduction.is referred as transduction.
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ElectroporationElectroporation Electroporation is technique involves electric Electroporation is technique involves electric
field mediated membrane permeabilization.field mediated membrane permeabilization.
It is based on the principle that high voltage It is based on the principle that high voltage pulses can induce cell plasma membrane fusepulses can induce cell plasma membrane fuse..
Electric shock induce cellular uptake of Electric shock induce cellular uptake of exogenous DNA from the suspending solution.exogenous DNA from the suspending solution.
It is simple & rapid technique for introducing It is simple & rapid technique for introducing genes in to the cells of various organisms.genes in to the cells of various organisms.
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WHY CLONE A GENE?
A particular gene can be isolated and its nucleotide sequence can be determined
Coding regions and control elements like promoters can be identified and analyzed
Protein/enzyme/RNA function can be investigated
Mutations can be identified, e.g. gene defects related to specific diseases
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Organisms can be ‘engineered’ for specific purposes, e.g. insulin production, insect resistance, etc.
If a protein is not abundant naturally, its gene can be cloned to produce the protein in large amounts
Cont….
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References References ::
Gene biotechnology by S. N. Jogdand.Gene biotechnology by S. N. Jogdand.
Biotechnology by U. Satyanarayana.Biotechnology by U. Satyanarayana.
Molecular Biology of Cell by Bruce Molecular Biology of Cell by Bruce Alberts.Alberts.
www.slide world.com.www.slide world.com.
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