Scribd
Upload
10 views10 pages

Recom Sel 1

The document discusses recombinant selection methods for screening libraries, which are classified into sequence-dependent cloning for unexpressed proteins and expression cloning for expressed proteins. It details various nucleic acid hybridization protocols, including plaque lift and colony hybridization, as well as the use of homologous and heterologous probes. Additionally, it covers techniques like Hybrid Release Translation (HRT) and Hybrid Arrest Translation (HART) for linking cDNA to proteins, and PCR methods for screening libraries.

Uploaded by

sou
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PPTX, PDF, TXT or read online on Scribd
10 views10 pages

Recom Sel 1

The document discusses recombinant selection methods for screening libraries, which are classified into sequence-dependent cloning for unexpressed proteins and expression cloning for expressed proteins. It details various nucleic acid hybridization protocols, including plaque lift and colony hybridization, as well as the use of homologous and heterologous probes. Additionally, it covers techniques like Hybrid Release Translation (HRT) and Hybrid Arrest Translation (HART) for linking cDNA to proteins, and PCR methods for screening libraries.

Uploaded by

sou
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PPTX, PDF, TXT or read online on Scribd

Recombinant selection

This involves screening of libraries

Broadly classified into

Methods when proteins are not expressed:-


sequence dependent cloning

Methods when proteins are expressed in the


library :- expression cloning
Nucleic Acid Hybridisation protocols

When proteins are not expressed:- dependent on


the nucleic acid sequence of the insert

• Plaque lift
• Colony hybridisation
• Differential screening and Subtractive
hybridisation
• Recombinational Probing
• HRT and HART
• PCR for screening libraries
The probe problem

Homologous v/s Heterologous probes : Heterologous


probing can be done if the gene in question is conserved

Degenerate probes :- Chemically synthesised if


information about the encoded protein or the partial
sequence of the gene is known. If the probe is made
from decoding the a a sequence of the protein some
allowance for codon degeneracy especially at the
wobble position should be given. Instead of a single
probe a collection of degenerate probes are used in this
case. Probes are 14-20 bases long

Abundancy probing ( generally for CDNA libraries)


Plaque Lift

Identical
prints of the
same library
can be made
and probed
with same or
different
probes
Colony
hybridisation
Recombinational Probing

This is done for library


screening by virtue of
homology betweeen the
probe maintained .
For this library is prepared in
a host containing vector
inserted with the probe.
If a homologous insert
sequence is found in the
phage vector; by virtue of
homologous recombination
will be inserted into the
phage vector. Selected by
supF mutation
Hybrid release translation (HRT) and
Hybrid Arrest translation (HART)

To link a cDNA to its corresponding protein

HRT : cloned cDNA is bound to a nitrocelulose


filter and hybridised with total cellular RNA or
unfractionated mRNA. Then the hybridised RNA
is eluted by heating in low salt buffer or
formamide. then the eluted mRNA is translated
and radio labelled polypeptides are analysed in
gel
HART :
mRNA will not direct protein synthesis when it is in
DNA:RNA hybrid.

mRNA preparation is mixed with a CDNA clone of


the gene in question. conditions are adjusted in
such a manner that RNA DNA hybridisation is
preferred. Then translation is done. Polypeptides
labelled with s adenosyl methionine is separated on
a gel and auto radiographed. The band which is
absent in the standard is the band of the
polypeptide encoded by the cloned cDNA.
Hybridisation is done in solution
PCR for screening libraries

Same limitation as that of Nucleic acid hybridisation.

A particular Clone is identified by flanking primers.

Instead of plating out as a library the clones are maintained in


multiwell plates and each plate is screened by PCR.

Positive wells are diluted and the process is repeated until a


single clone is obtained

Use of degenerate primers: unknown members of a gene family


or homologs from other species

Some mismatches should be allowed but avoid mismatch at the


3, nucleotide
References:

Old and Primrose, Principles of


Gene Manipulation 5th and 6th
editions

Thank you

You might also like