biotechnology

1. SYNTHESIS OF cDNA
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.
AAAAAAAAAAn5’ cap

5. 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.

7. 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.

8. (1) SCREENING BY HYBRIDIZATION
1. 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.
2. Membrane is coated with replica of the microbial colonies and the
recombinant 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 short
synthetic oligonucleotide probes.
6. Excess probes are washed off.
7. Colonies containing recombinant clones complementary to the
probe appear as a dot on the filter.
8. 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.
9. 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.

11. 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.

12. (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.

13. 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.

14. 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.

15. 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).

16. 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.

17. 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.