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 treatment compared to 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
Expression cloning
Done when the insert is able to express the
protein encoded as a fully functional one or as a
fusion with a tag poly peptide
Methods include
• Genetic selection
• Immunochemical screening
• South Western and North Western Screening
• Functional cloning
1. Functional complementation
2. Gain of function
Genetic Selection
1. Presence of vectors are selected by using
antibiotic resistance markers. For eg. Ampicillin
resistance marker in pUC
2. Selection of inserted sequences
Eg. 1. Methotrexate resistant mouse DHFR cloned
in bacterial cells. Selection is done by Methotrexate
(gain of function)
2. auxotrophic mutants are selected by
reversion to autotrophs by the inserted DNA.
eg. Yeast his gene insert selected by reversion of a
histidine auxotrophic bacteria to his autotroph
(functional complementation)
Immunochemical screening
Depends on Three points
1.A polyvalent antisera contain different IgG
molecules which can bind different
determinants on the antigen
[Link] molecules can strongly bind plastics
such as polyvinyl from which they are not
removed by washing
3. IgG can be easily labelled by I125 in vitro
Transformed cells are plated on a
petri plate
Cells are lysed
1. By chloroform vapour
2. By Spraying an aerosol of virulent
phages
3. By inducing temp sensitive
prophage
4. Polyvinyl coated with unlabelled
antibody is applied
5. Protein is lifted
6. Second labelled IgG is applied
2
1
South western screening/ North Western Screening
Identifying clones expressing fusions of nucleic acid
binding proteins
Nitrocellulose plaque lifted proteins are identified by a
labelled duplex DNA probe/ RNA
Depends on two factors
1. The binding activity should be by a single
polypeptide
2. Activity should be present in fusion polypeptide form
Eg. Identification of transcription factors
Functional cloning
Depends on the full functionality of the protein
[Link] complementation
Eg. 1. Yeast his gene cloning in E coli
2. Identification Shaker- 2 mouse mutation
and human homologue DFNB3 ( deafness
gene)
2. Gain of function . Eg. Cloning of Mouse dhfr,
cloning of oncogenes in 3T3 fibroblast or in
nude mice
Difference cloning
1. Difference cloning with DNA libraries
• Differential screening
• Subtractive hybridisation
2. Difference cloning by PCR
• Differential display PCR and arbitrarily primed PCR
• Representational Difference Analysis
Differential screening &
Subtractive hybridisation
These techniques are adopted
for cloning differentially
expressed mRNAs
Subtractive hybridisation
Is done identifying those genes
that are expressed when a
particular treatment, stage or
tissue is analysed.
Recently much interest is there
for these technique with the
advent of DNA microarrays
Enriching Differences- Subtractive cloning
Used to create a cDNA library enriched in a particular type of
sequences.
Eg: Cloning of Transcripts expressed in rat Dentate Gyrus( DG)
by Kainate treatment (a glutamate analogue inducing seizures
and memory related changes).
First strand cDNA was synthesised from the mRNA of kainate
treated animals.
Ubiquitous mRNA were negated by hybridising with an excess
of biotynylated mRNA isolated from untreated rat Brain.
Hybrids and unbound mRNA are removed by Streptavidin
column.
Unhybridised cDNA (Kainate specific) are made double
stranded and cloned to get an enriched library.
• Differential display PCR and arbitrarily primed PCR
• Advantages
• Speed, can be done even with small amount of starting material
• An mRNA fingerprint is developed by amplifying mRNA from two
sources and running them side by side revealing differentially
expressed genes
• In differential display PCR first strand is synthesised with an
oligodT primer with 2bp extensions
• In AP PCR antisense primer is arbitrary and can bind anywhere
along the mRNA
• In both cases an arbitrary primer is used for second strand
synthesis. Differentially expressed cDNA can be excised from the
gel and further characterised
Class Assignment
Representational Difference Analysis
References
Old and Primrose of gene Manipulation
5th and 6th edns
Thank You
