C dna library

A DNA library prepared from m RNA and useful to explore new gene and other tissues specific gene expression

1. Presenting
C DNA
LIBRARY

2. Definition
•Library means collection and c DNA library is
collection of c DNA corresponding to all the mRNA
being formed in an organisms

3. OVERVIEW OF C DNA
LIBRARY

4. Steps
Isolation
Synthesis of C DNA
Cloning

5. Steps of C DNA library preparation

6. Isolation of total RNAs
A cell contains all types of RNA . The first steps is to isolate total RNAS
•Sample in 1 ml Trizolereagent
•Homogenize
•Add 200 micro l chloroform
•Vortex for 2 minutes
•Incubate in icebox for 15 min.
•Centrifuge to get phase separation at 12000 g at 4 deg c for 10 ‘
•Collect red aqphase in ependrof
•Pptthe RNA by mixing with 0.5 ml isopropanol
•Incubate in ice box for 10’
•Centrifuge at 4 deg C for 10’
•Remove supernatant
•wash pellet with 1ml 70% ALCOHAL by flicking
•Centrifuge at 7500 g at 4 deg for 10 ‘
•Remove supernatant
•DisolveRNA in the RNASefree water .

7. Isolation of m RNA from total RNA
:
••Because most eukaryotic mRNAs carry 3′-poly(A) tails, mRNA
can be selectively isolated from preparations of total cellular RNA
by oligo(dT)-cellulose chromatography.
•DNA copies of the purified mRNAs are synthesized by first
annealing short oligo (dT) chains to the poly(A) tails.
the poly-A tail isshortened (presumably during translation) by
the CCR4-NOT and PARN complexes.
As the poly-A shortens, there are less PABP that can associate
with the eIF4F.
Once the poly-A is short enough, the eIF4F-cap complex is less
stable, leading to successful recruitment of the decapping
complex to the cap.

8. Isolation of m RNA
•Isolation Procedure:
•Add 50 μl of 5M NaClper450 μl of cell lysateor total RNA
solution or dissolve total RNA sample in 450 μl of Elution
Buffer then add 50 μl of 5M NaCl.
•Make sure that sample is totally dissolved.
•If not, microcentrifugefor 5 minutes to pellet insoluble
material.
•The mRNA can be isolatedfrom total RNA by oligo(dT)
chromatography. ... One of most convincing and reliable
methods of mRNA isolation is the magnetic separation
method with oligo(dT) bound on the surface of
paramagnetic beads. A protocol for isolation of poly A +
RNA directly from cell lysateis described below.

9. Column affinity chromatography for m
RNA isolation

10. Protocol for isolation of m RNA

11. II step -C DNA synthesis
••These oligo(dT) chains serve as primers for
reverse transcriptase-driven synthesis of DNA
Reverse transcriptase is an enzyme that
synthesizes a DNA strand, copying RNA as the
template.
•DNA polymerase is then used to copy the
DNA strand and form a double-stranded
(duplex DNA) molecule.

12. C DNA Synthesis

13. Gubber Hoffman method of C DNA synthesis

14. Cloning of C DNA for Library
••Lastly Linkers are added to the DNA duplexes rendered from the mRNA templates,
and the cDNA is cloned into a suitable vector.
••Once a cDNA derived from a particular gene has been identified, the cDNA
becomes an effective probe for screening genomic libraries for isolation of the gene
itself.
••Because different cell types in eukaryotic organisms express selected subsets of
genes, RNA preparations from cells or tissues in which genes of interest are
selectively transcribed are enriched for the desired mRNAs.
•• cDNA libraries prepared from such mRNA are representative of the pattern and
extent of gene expression that uniquely define particular kinds of differentiated
cells.
.
Important vectors -λgt10 & λgt11 Phage vectors:
Insertion Vectors-Accept Approximately 7.6 kb & 7.2 kb
λZAP series:Phage clones have to be sub cloned back into plasmid
called Phasmids Advantages: High capacity (up to 10Kb)
Phage-λ vectors were mostly used for cDNAcloning & expression

15. b. IMPROVED METHOD OF C DNA CLONING

16. b. Homo-polymer tailing method

17. Screening of c DNA

18. Plaque Lift Methods

19. Immunological screening methods

20. Importance of c DNA library
•C DNA can not be prepared from m RNA as it is very unstable and
vulnerable .
•Genomic libraries are easier to prepare that contains all the
genomic sequences
•C DNA library are useful for eukaryotic gene expression
•Condensed protein encoded by gene library have more junk DNA
sequence while C DNA has less no junk DNA sequences
•C DNA has no intron s so it can be expressed in e .coli directly
•Are very useful to identify the genes
•Differential , tissues specific gene can be studied
•

21. Advantage and disadvantage
•Advantage: Size of cDNA clone is significantly
lower than the Genomic DNA library
Disadvantage:Cleavage with S1 nuclease
results in loss of certain amount of sequence
at the 5’ end of the clone