1. Speaker
P.RAMESH
Ph.D (ABC)

2. INTRODUCTION
 In a simple cloning procedures, DNA fragments are
joining to vector followed by introduction into the host
cells & selection/screening will be done
 If a source of a donor DNA is very complex???
Genomic DNA
c DNA
 Cloning strategy:
DNA
fragment
Joining to
Vector
Host cell
Selection/
screening

3.  Two major approaches for isolating sequences from
complex sources (Genomic DNA/cDNAs)
Cell based cloning strategy:
It is to divide the source of DNA into fragments &
clone everything
Such a collection of clones representation of entire
population called Library
Screen the Library:To identify our clone of interest
using a procedures that discriminates between the
desired clone & all others

4. Cont…
Target sequence by PCR:
Selectively amplify the target sequences directly
from source of DNA using PCR & cloned
In PCR approach, screening step is built into 1st
stage of the procedure
So that only selected fragments are actually cloned

5. Complementary DNA (cDNA)
Libraries
 cDNA Library is a population of mRNAs
 Generated by Reverse Transcription of cellular mRNA,
reveals expression profiles in different cell types and
developmental stages
 Cloned eukaryotic cDNAs have their own special uses
 Advantage: Size of cDNA clone is significantly lower
than the Genomic DNA library

6. Cont…
 Phage-λ vectors were mostly used for cDNA cloning &
expression
 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)

7. Early cDNA cloning Strategy
3 Major Steps:
First-strand DNA synthesis on the mRNA by Reverse
transcriptase
Removal of mRNA template
Second-strand DNA synthesis using 1st strand DNA as
a template by DNA pol-I
 cDNA cloning was based on the Homo-polymer tailing
method (1970s)

8. Fig: An Early Cloning Strategy

9. Fig: Homo-polymer Tailing Method

10. Cont…
 Disadvantage:
Cleavage with S1 nuclease results in loss of certain
amount of sequence at the 5’ end of the clone
 Other Methods:
Improved method by cDNA cloning
(Land et al., 1981)
Directional cloning
Non-directional cloning (Gubbler-Hoffman method)
CAPture method
 Oligo-cappling method

11. Fig: Improved Method for cDNA cloning

12. Fig: Directional cDNA cloning method

13. Fig: Gubbler-Hoffman method

14. Cont…
 Drawbacks:
Generally 3’end bias in the resulting library
Native enzymes have poor processivity & intrinsic
RNase activity
Optimal activity for native enzymes at 370C

15. Fig: CAPture Method for full length cDNA

16. Fig: Oligo-capping Method

17. Screening of cDNA Libraries
Colony Hybridization
N.Acids Hybridization
Plaque-Lift Method
Immunological Screening

18. Fig: Colony Hybridization Method

19. Fig: Plaqe lift Method

20. Fig: Immunological screeningMethod

22. Fig: Linear map of λZAP Phasmid vector