Date post: | 15-Dec-2014 |
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Education |
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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