Speaker

P.RAMESH

Ph.D (ABC)

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

Selection/screening

Joining to Vector Host cell

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

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

Cont…

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

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)

Cont…

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)

Fig: An Early Cloning Strategy

Fig: Homo-polymer Tailing Method

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

Directional cloning

Non-directional cloning (Gubbler-Hoffman method)

CAPture method

Oligo-cappling method

Cont…

(Land et al., 1981)

Fig: Improved Method for cDNA cloning

Fig: Directional cDNA cloning method

Fig: Gubbler-Hoffman method

Drawbacks:

Generally 3’end bias in the resulting library

Native enzymes have poor processivity & intrinsic

RNase activity

Optimal activity for native enzymes at 370C

Cont…

Fig: CAPture Method for full length cDNA

Fig: Oligo-capping Method

Screening of cDNA Libraries

Colony Hybridization

Plaque-Lift Method

Immunological Screening

N.Acids Hybridization

Fig: Colony Hybridization Method

Fig: Plaqe lift Method

Fig: Immunological screeningMethod

Fig: Linear map of λZAP Phasmid vector