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)
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