SYNTHESIS OF cDNA

Presented by: DEEPTI SINGH

Ph.D. (BIOTECHNOLOGY)

First Semester

Enrol. No. B-1384/14

COLLEGE OF BIOTECHNOLOGY

DUVASU, MATHURA

INTRODUCTION

cDNA library is a population of bacterial transformants or phage

lysates in which each mRNA isolated from an organism or tissue

is represented as its cDNA insertion in a plasmid or a phage

vector.

It is absolutely essential when the expression of an eukaryotic

gene is required in prokaryote.

Produced by using mRNA as a template.

DNA copy of RNA molecule is produced by the enzyme reverse

transcriptase(RNA dependent DNA polymerase).

This enzyme performs similar reactions as DNA polymerase.

Oligonucleotide primer is required which is annealed with the

mRNA molecule with the poly A tail at the 3’end.

mRNA ISOLATION

• Most eukaryotic mRNAs are polyadenylated at

their 3’ ends

• oligo (dT) can be bound to the poly(A) tail

and used to recover the mRNA.

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SYNTHESIS OF cDNA

1. Obtain mRNA. Poly(A)+mRNA is especially essential for this

because the poly A tail binds to the oligo-dT DNA.

2. Copy mRNA with the enzyme reverse transcriptase.

3. Degrade mRNA with base.

4. Make double stranded cDNA using the mRNA as template, DNA

polymerase from E.coli, and deoxyribonucleoside triphosphate

as substrates. A hairpin loop at the end of the cDNA is formed by

reverse transcriptase, may serve as primer.

5. Treat with S1 nuclease to remove the hairpin loop.

6. The resulting double stranded DNA is ligated into the cloning

vector.

7. The double stranded cDNA may be used as a probe in

hybridization.

LIBRARY SCREENING

Clone containing the desired DNA fragment must be

isolated.

It may be done by DNA hybridization on a nylon or

nitrocellulose membrane or by examining clones for protein

expression.

Probes may be derived from known DNA of closely related

organisms or synthesized in the laboratory.

(1) SCREENING BY HYBRIDIZATION

1. Colonies of yeast or bacteria or phage plaques on a bacterial lawngrown on solid medium in a petridish are transferred to a nylon ornitrocellulose membrane by laying it on top of colonies.

2. Membrane is coated with replica of the microbial colonies and therecombinant DNA molecule it contains, when it is lifted off.

3. Some of the microbial colony remains on the agar.

4. Colony on the membrane are lysed.

5. DNA molecules are denatured and hybridized to DNA,RNA or shortsynthetic oligonucleotide probes.

6. Excess probes are washed off.

7. Colonies containing recombinant clones complementary to theprobe appear as a dot on the filter.

8. Filter is the replica of the original plate so the location of the colonycontaining the desired insert DNA can be identified and the clonesubcultured.

9. Colony is grown in larger volumes of medium, tested again to besure the DNA of interest is present, then used to replicate largequantities of the desired DNA.

IDENTIFICATION OF A SPECIFIC CLONE FROM

A PHAGE LIBRARY BY MEMBRANE

HYBRIDIZATION TO A RADIOLABELED PROBE

The position of the signal on the autoradiogram identifies

the desired plaque on the plate.

In practice, in the initial plating of a library the plaques

are not allowed to develop to a visible size so that up to

50,000 recombinants can be analyzed on a single plate.

Phage particles from the identified region of the plate are

isolated and replated at low density so that the plaques

are well separated.

Then pure isolates can be obtained by repeating the

plaque hybridization as shown in the figure.

(2) SCREENING BY ASSAY

1. Done phenotypically by looking for visual expression of a

trait or for enzyme activity.

2. Expressed protein may also be detected using an

immunological assay.

3. Antibody screening is carried out in a manner similar to

western blotting using nitrocellulose membrane as

described above.

4. Colonies of microbes on the filter are lysed and then

probed with a primary antibody that binds to the protein of

interest.

5. The antibody is detected with a secondary Ab, one that

binds to the primary Ab and is visualized in a calorimetric

enzyme assay.

6. Positive spots, correlated to the master plate, are used to

pick colonies containing recombinant clones of interest.

ADAPTER

An adapter in genetic engineering is a short, chemically

synthesized, double stranded DNA molecule which is used to link

the ends of two other DNA molecules.

It may be used to add sticky ends to cDNA allowing it to be

ligated into the plasmid much more efficiently.

Adapters are used to link the ends of two DNA molecules that

have different sequences at their ends.

A conversion adapter is used to join a DNA insert cut with

one Restriction enzyme, say EcoRl, with a vector opened with

another enzyme, Bam Hl.

This adapter can be used to convert the cohesive end produced

by Bam Hl to one produced by Eco Rl or vice versa.

GENE CASSETTE

A gene cassette is a type of mobile genetic element that

contains a gene and a recombination site.

They may exist incorporated into an integron or freely as

circular DNA.

Gene cassettes often carry antibiotic resistance genes.

An example would be the kanMX cassette which

confers kanamycin (an antibiotic) resistance upon bacteria.

In genetic engineering, a gene cassette refers to a

manipulable fragment of DNA carrying, and capable of

expressing, one or more genes of interest between one or

more sets of restriction sites.

It can be transferred from one DNA sequence (usually on

a vector) to another by 'cutting' the fragment out

using restriction enzymes and 'pasting' it back into the new

context.

INTEGRONS

Are genetic structures in bacteria which express and are

capable of acquiring and exchanging gene cassettes.

These cassettes typically carry a single gene without a

promoter.

The entire series of cassettes is transcribed from an adjacent

promoter.

The gene cassettes are speculated to be inserted and excised

via a circular intermediate.

This would involve recombination between short sequences

found at their termini and known as 59 base elements.

The 59-be are a diverse family of sequences that function as

recognition sites for the site-specific integrase (enzyme

responsible for integrating the gene cassette into an integron).

LINKERS

Polylinker (also known as a multiple cloning site or simply a linker),

a short segment of DNA with many restriction sites; commonly used

in biotechnology, bioengineering, and molecular genetics.

Linker proteins (or adaptor proteins), proteins that provide

mechanisms by which receptors can amplify and regulate

downstream effector proteins, e.g.:

Linker of activated T cells, a protein in the biochemical signaling

path transferring signals from T cell antigen receptors.

B-cell linker, a human gene that encodes a linker protein related

to B cells.

Linker DNA, the part of a genomic DNA strand that connects two

nucleosomes.

REFERENCES

http://en.wikipedia.org/w/index.php?title=Adapter_(genetics)&oldid=6016

47985.

Hall, RM; Collis, CM (1995). "Mobile gene cassettes and integrons:

Capture and spread of genes by site-specific recombination". Molecular

microbiology 15 (4): 593–600. PMID 7783631.

Scheppler, J.A., Cassin,P.E. and Gambier, R.M., Biotechnology

explorations, (2002): 259-262.

Purohit, S.S., Biotechnology, (2005): 62-65.

Singh, B.D., Biotechnology, (2010).

Methods Molecular Biology, (2008);410:55-80.