Recombinant DNA technology

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RECOMBINANT DNA TECHNOLOGYPresenter : saranya . S Moderator : dr.v.balasubramaniyan 1

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DNA CLONINGDNA cloning protocolPlasmid cloningBacteriophage cloningYeast artificial chromosome

2Recombinant DNA libraries

Genomic librarycDNA librarySummary

Molecular cloning / DNA cloningMolecular cloning refers to process of making multiple copies of DNA 3Step 1 : Fragmentation breaking apart a strand of DNAStep 2 : ligation gluing together pieces of DNA in a desired sequenceStep 3 : transfection inserting the newly formed DNA in to cellsStep 4: screening / selection selecting out the cells that were successfully transfected with the new DNA

DNA Cloning Protocol4PLASMID CLONING STRATEGY

Recombinant DNA Cloning Procedure5Foreign DNArDNAHost cellDesired productsAntibiotics Antibodies Blood factorsHormones Enzymes VaccineFine chemicalsPlasmid DNA

STEP 1. RE DIGESTION OF DNA SAMPLE6

STEP 2. RE DIGESTION OF PLASMID DNA

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STEP 3. LIGATION OF DNA SAMPLE AND PLASMID DNA8

STEP 4. TRANSFORMATION OF LIGATION PRODUCTSThe process of transferring exogenous DNA into cells is called transformation

Chemical method utilizing CaCl2 and heat shock to promote DNA entry into cells.

Electroporation based on a short pulse of electric charge to facilitate DNA uptake.9

CHEMICAL TRANSFORMATION WITH CALCIUM CHLORIDE

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TRANSFORMATION BY ELECTROPORATION11

STEP 5. GROWTH ON AGAR PLATES12

STEP 5 : Blue-white ScreeningBlue colonies represent Ampicillin-resistant bacteria that contain vector and express a functional alpha fragment from an intact LacZ alpha coding sequence

White colonies represent Ampicillin-resistant bacteria that contain Insert and do not produce LacZ alpha fragment13

-Galactosidase Activity Can Be Used As An Indicator Of The Presence Of A Foreign DNA Insert14

OVERVIEW

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Amplification and purification of recombinant plasmid DNAPositive colony containing recombinant plasmid DNA is transferred aseptically to liquid growth medium, the cells will continue to multiply exponentially

Within a day or two, a culture containing trillions of identical cells can be harvested

Plasmid DNA can be purified from crude cell lysates by chromatography (using silica gel or anion exchange resins)17

Amplification and purification of recombinant plasmid DNAThe purified plasmid DNA can then be eluted and recovered by ethanol precipitation in the presence of monovalent cations

Ethanol precipitation of plasmid DNA from aqueous solutions yields a clear pellet that can be easily dissolved in an appropriate buffered solution18

Bacteriophage Lambda () Cohesive Sites19

Bacteriophage Lambda () Cohesive Sites20

Yeast Artificial Chromosome

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Red white screeningSUP4 encodes a tRNA that suppresses the Ade2-1 UAA mutationADE1 and ADE2 encode enzymes involved in the synthesis of adenine (phosphoribosyl amino-imidazole-succinocarbozamide synthetase and phosphoribosylamino-imidazole carboxylase).In the absence of these critical enzymes, Ade2-1 mutant cells produce a red pigmentBut Ade2-1 mutant cells expressing SUP4 are white because the Ade2-1 mutation is suppressedRed colonies contain recombinant YAC vector DNAWhite colonies contain non recombinant YAC vector DNA.22

Recombinant DNA LibrariesGenomic library

cDNA library

Chromosomal library23

Creation of a Genomic DNA library using the phage- vector EMBL3A24

# High-molecular-weight genomic DNA is partially digested with Sau3AI

# The vector is digested with BamHI and EcoRI, which cut within the polylinker sites

# The vector arms are then ligated with the partially digested genomic DNA

# The only package able molecules are recombinant phages. These are obtained as plaques on a P2 lysogen of sup+ E. coli

# The Spi selection ensures recovery of phage lacking red and gam genes25

cDNA Libraries A cells mRNA molecules can be copied to make complementary DNA strands (cDNA)

The cDNA derives only from mature mRNA. Introns are not present

The poly(A) tail at the 3 end of the mRNA is useful for:Isolating mRNA from cell lysates by passage over an oligo(dT) columnPriming the synthesis of cDNA, by providing a known 5 sequence26

An Early cDNA Cloning Strategy, Involving Hairpin Primed Second-strand DNA Synthesis And Homopolymer Tailing To Insert The cDNA Into The Vector27

An Early cDNA Cloning Strategy, Involving Hairpin Primed Second-strand DNA Synthesis And Homopolymer Tailing To Insert The cDNA Into The Vector28

Improved Method For cDNA Cloning. The First Strand Is Tailed With Oligo(dc) Allowing The Second Strand To Be Initiated Using An Oligo(dg) Primer29

Oligo-dG, Reverse transcriptase + 4 dNTPsInsert in to vector by either homopolymer tailing or linkers

Summary 30Gene cloning

vectorsHost cellPreparation of cDNA library# Orgin of replication# Suitable marker# Single restriction# Expression signalPlasmidsPhage Prokaryote Eukaryote # transformation #transfection # physical method#chemical methodGenomic librarycDNA libraryCan be cloned for further use

Inserted in to host cell

References Sambrook, J., Russell, D.W. (2001) Molecular Cloning: a Laboratory Manual, 3rd edn. Cold Spring Harbor Laboratory Press, New YorkAusubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., Struhl, K., eds (2002) Short Protocols in Molecular Biology, 5th edn. John Wiley & Sons, New YorkLodge j., lund P., Gene cloning principles and application 2nd edition Primrose SB., Twyman RM., Principles of gene manipulation and genomicsBrown TA., Gene cloning and DNA analysis., 6th editionRecombinant DNA technology and DNA cloning., chapter 8Molecular cloning technical guide

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THANK YOU 32