1

RECOMBINANT DNA

TECHNOLOGYPRESENTER : SARANYA . S MODERATOR : DR.V.BALASUBRAMANIYAN

2DNA CLONING DNA cloning protocol Plasmid cloning Bacteriophage λ

cloning Yeast artificial

chromosome

Recombinant DNA libraries

Genomic librarycDNA library

Summary

3Molecular cloning / DNA cloning

Molecular cloning refers to process of making multiple copies of DNA

Step 1 : Fragmentation – breaking apart a strand of DNA

Step 2 : ligation – gluing together pieces of DNA in a desired sequence

Step 3 : transfection – inserting the newly formed DNA in to cells

Step 4: screening / selection – selecting out the cells that were successfully transfected with the

new DNA

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DNA Cloning ProtocolPLASMID CLONING

STRATEGY

5Recombinant DNA Cloning Procedure

Foreign DNA

rDNA Host cell

Desired product

sAntibiotic

s Antibodies Blood factors

Hormones Enzymes Vaccine

Fine chemical

s

Plasmid DNA

6STEP 1. RE DIGESTION OF DNA SAMPLE

7STEP 2. RE DIGESTION OF PLASMID DNA

8STEP 3. LIGATION OF DNA SAMPLE AND PLASMID DNA

9STEP 4. TRANSFORMATION OF LIGATION PRODUCTS

The 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.

10CHEMICAL TRANSFORMATION WITH

CALCIUM CHLORIDE

11TRANSFORMATION BY ELECTROPORATION

12STEP 5. GROWTH ON AGAR PLATES

13STEP 5 : Blue-white

Screening Blue 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 fragment

14β-Galactosidase Activity Can Be Used As An Indicator Of The Presence Of A Foreign DNA

Insert

15OVERVIEW

16

17Amplification and purification of recombinant plasmid DNA

Positive 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)

18Amplification and purification of recombinant plasmid DNA

The 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 solution

19Bacteriophage Lambda (λ) Cohesive Sites

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Bacteriophage Lambda (λ) Cohesive Sites

21Yeast Artificial Chromosome

22Red white screening SUP4 encodes a tRNA that suppresses the Ade2-1

UAA mutation ADE1 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 pigment

But Ade2-1 mutant cells expressing SUP4 are white because the Ade2-1 mutation is suppressed

Red colonies contain recombinant YAC vector DNA White colonies contain non recombinant YAC

vector DNA.

23Recombinant DNA Libraries

1. Genomic library

2. cDNA library

3. Chromosomal library

24Creation of a Genomic DNA library using the phage-λ

vector EMBL3A# 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

25# 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 genes

26cDNA Libraries

A cell’s 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:

1. Isolating mRNA from cell lysates by passage over an oligo(dT) column

2. Priming the synthesis of cDNA, by providing a known 5’ sequence

27An Early cDNA Cloning Strategy, Involving Hairpin Primed Second-strand DNA Synthesis And Homopolymer Tailing To Insert The cDNA

Into The Vector

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An Early cDNA Cloning Strategy, Involving Hairpin Primed Second-strand DNA Synthesis And

Homopolymer Tailing To Insert The cDNA Into The Vector

29Improved Method For cDNA Cloning. The First Strand Is Tailed With Oligo(dc) Allowing The

Second Strand To Be Initiated Using An Oligo(dg) Primer

Oligo-dG, Reverse

transcriptase + 4 dNTPs

Insert in to vector by either homopolymer

tailing or linkers

30Summary Gene cloning

vectorsHost cell

Preparation of cDNA library

# Orgin of replication

# Suitable marker# Single

restriction# Expression

signal

Plasmids

Phage

Prokaryote

Eukaryote

# transformation #transfection

# physical method#chemical method

Genomic library

cDNA library

Can be cloned for further useInserted

in to host cell

31References Sambrook, J., Russell, D.W. (2001) Molecular Cloning: a Laboratory

Manual, 3rd edn. Cold Spring Harbor Laboratory Press, New York Ausubel, 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 York

Lodge j., lund P., Gene cloning principles and application 2nd edition Primrose SB., Twyman RM., Principles of gene manipulation and

genomics Brown TA., Gene cloning and DNA analysis., 6th edition Recombinant DNA technology and DNA cloning., chapter 8 Molecular cloning technical guide

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