Transcript Library screening

Library screening
 Heterologous and homologous gene probes
 Differential screening
 Expression library screening
Heterologous and
homologous gene probes
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A genomic or cDNA library whether in plasmid or in
a lambda vector contains a large number of
different recombinants
There are number of methods for screening
libraries for specific sequences
The basis of the procedure is the ability of two
stranded DNA molecules of complementary or nearly
complementary sequence to form a duplex
Probe (a labeled single stranded DNA molecule)
Homologous: identical sequences
Heterologous: not identical sequences
Heterologous and
homologous gene probes
 The ability of heterologous sequence to
form duplexes depends on:
1. The length of matching sequence
2. Base composition
3. The number of mismatches
4. The reaction (hybridization) conditions
5. The stringency of the condition in which the
probe is washed after hybridization
Type of probe
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The probe may be a sequence which has already
been cloned, such as:
cDNA
PCR product
Genomic fragment from a different species
An incomplete part of the gene or a cDNA to be
isolated
A sequence from a different gene from the same
species
A synthetic oligonucleotide
Mixed sets of sequences derived from particular
mRNA population
Procedure
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Plates are prepared with either bacterial colonies containing a
plasmid library or with plaques from a phage library growing on
bacteria
Colonies are replicated on nitrocellulose or other hybridization
filter and grown further, whereas plaques are lifted onto
filter and processes immediately
The filter are processed to lyse the bacterial cells or to
break open (disrupt) the phage protein coat and denature and
fix the DNA in situ
There are then probed with a labeled DNA fragment which
will hybridize to the sequence of interest
The position of hybridization signal is determined by
autoradiography and used to identify the colony or plaque
containing the sequence
The colony or plaque is picked from the master plate and
replate at a density to give individual colonies or plaques and
screened again
Screening a library by hybridization
Application
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Isolation of homologous gene sequences from the
same species (use of a cDNA probe to isolate a
genomic clone, use of PCR product to isolate cDNA
or genomic clones, use of a partial gene or cDNA
sequence to isolate a full-length sequence
Identification of closely related gene in a gene
family
Isolation of related genes from other species
Isolation of genes encoding proteins which have
been completely or partially sequence. The protein
sequence is back-translated to give a DNA sequence
and it is used to design an oligo-nucleotide probe
Differential screening
 The isolation of NA sequences from a
library by means of differential
hybridization with complex NA probes
 The principle is based on differences in the
concentration of NA species between two or
more samples
 mRNas are the NA sequences studied
 Differential screening aims at isolating
differentially transcribed mRNA
 Differential screening represents a means
to isolate NA sequences on the basis of a
common regulatory mechanism rather than
their identities or function
Differential screening
 Differential screening has been used to
identify cDNA clones that reflect mRNAs
present at different level in different cell
types
 It is a technique that allows the analysis of
differentially regulated gene transcription
and the cloning of mRNAs which are
differentially transcribed between two or
more samples
 Samples may be different cell types,
tissues, organs, the same cells which have
been subjected to different treatment,
cells which have a different metabolic or
physiological status
Methodology
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Cellular RNA is extracted from samples
Poly(A) RNAs are then purified from the total RNAs and
poly(A) RNA fraction obtained from one sample is used as a
template for the synthesis of the corresponding cDNA, which
is then cloned into vector
The cDNA library is then plated at a relatively low density to
facilitate subsequent identification of individual clones by
colonies or plaques hybridization
Two replica filters are taken from the master plate and
hybridized independently to labeled probes obtained by
reverse transcription of the mRNA fraction
After autoradiography, clones which show different
intensities of hybridization signal with the two probes
identify mRNas for differentially transcribed genes
The clones are then selected from master plate and usually
subjected to a second round of differential hybridizaton to
confirm the results obtained in the first round and eliminated
the artefacts
The selected clones are characterized
Expression library screening
 Rapidly screen large cDNA libraries
expressing foreign proteins using antibody
probes
 It is enable us to isolate genes expressing
certain proteins against which antibodies
could be raised
 A number of factors can influence the
successful outcome of screening cDNA
expression libraries:
1. The protein one is looking for must be
expressed in the tissue which is used to
extract the RNA needed for the
construction of the cDNA library
2. The quality of antibody itself
Screening a library with antibodies