Synthesis of c dna

biotechnology

1. SYNTHESIS OF c DNA Presented by: DEEPTI SINGH Ph.D. (BIOTECHNOLOGY) First Semester Enrol . No. B-1384/14 COLLEGE OF BIOTECHNOLOGY DUVASU, MATHURA

2. 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.

3. 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. AAAAAAAAAAn 5’ cap

4. SYNTHESIS OF c DNA Obtain mRNA. Poly(A)+mRNA is especially essential for this because the poly A tail binds to the oligo-dT DNA. Copy mRNA with the enzyme reverse transcriptase. Degrade mRNA with base. 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. Treat with S1 nuclease to remove the hairpin loop. The resulting double stranded DNA is ligated into the cloning vector. The double stranded cDNA may be used as a probe in hybridization.

5. 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.

6. (1) SCREENING BY HYBRIDIZATION Colonies of yeast or bacteria or phage plaques on a bacterial lawn grown on solid medium in a petridish are transferred to a nylon or nitrocellulose membrane by laying it on top of colonies. Membrane is coated with replica of the microbial colonies and the recombinant DNA molecule it contains, when it is lifted off. Some of the microbial colony remains on the agar. Colony on the membrane are lysed . DNA molecules are denatured and hybridized to DNA,RNA or short synthetic oligonucleotide probes. Excess probes are washed off. Colonies containing recombinant clones complementary to the probe appear as a dot on the filter. Filter is the replica of the original plate so the location of the colony containing the desired insert DNA can be identified and the clone subcultured . Colony is grown in larger volumes of medium, tested again to be sure the DNA of interest is present, then used to replicate large quantities of the desired DNA.

7. 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.

8. (2) SCREENING BY ASSAY Done phenotypically by looking for visual expression of a trait or for enzyme activity. Expressed protein may also be detected using an immunological assay. Antibody screening is carried out in a manner similar to western blotting using nitrocellulose membrane as described above. Colonies of microbes on the filter are lysed and then probed with a primary antibody that binds to the protein of interest. The antibody is detected with a secondary Ab , one that binds to the primary Ab and is visualized in a calorimetric enzyme assay. Positive spots, correlated to the master plate, are used to pick colonies containing recombinant clones of interest.

9. 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.

10. 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.

11. 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 ).

12. 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 .

13. REFERENCES http://en.wikipedia.org/w/index.php?title=Adapter_(genetics)& oldid=601647985. 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 .

14. Thank you.