This document discusses different strategies for cloning DNA fragments from complex sources like genomic DNA or cDNA. There are two major approaches - cell-based cloning, which divides the DNA into fragments that are cloned to create a library, and directly amplifying target sequences using PCR. The document focuses on cDNA library construction, explaining that cDNA libraries reveal gene expression profiles. It describes early cDNA cloning methods and their limitations, as well as improved directional and non-directional cloning techniques. Finally, it discusses various screening methods for identifying clones of interest from cDNA libraries, including colony hybridization, plaque lifts and immunological screening.

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