Gene Cloning

Screening DNA Libraries 133
probe in a hybridization experiment to screen the petal library. Those
clones detected by the leaf cDNA probes will represent genes found in both
tissues. The few that are not detected will be genes expressed in the petal
and not in the leaf; your gene of interest should be one of these. This is a
very powerful tool and has been used to examine which genes are
expressed in different tissues or at different stages of development. This
approach has been combined with modern micro-array technology in the
development of a new area of study called transcriptomics, which is discussed
in detail in Section 11.6.
A further refinement of differential screening that can been used to
identify genes expressed under specific conditions, but not in control conditions,
is the construction of a subtractive library. In a subtractive library,
cDNAs common to both the test and control conditions are eliminated,
leaving only those cDNAs which are specific to the test conditions. This is
usually achieved by hybridization of cDNA from the two populations and
elimination of the double-stranded molecules, which represent sequences
present in both samples. Because a subtractive library is enriched for the
target sequences it can be particularly effective in identifying genes which
are expressed at low levels.
5.10 Using PCR to Screen a Library
If you were to extract DNA from a clone from a library and use it as the template
in a PCR reaction using specific primers, and you were to get a product
of the size you expected, that would be a way of demonstrating that the
clone most probably contained the specific DNA sequence of interest. This
principle has been developed into a method for library screening. The
method involves performing PCR reactions on pools of clones to reduce
the total number of PCR reactions which would be needed to screen every
clone in a library.
In order to adapt PCR to screening a library, without having to perform
a separate PCR reaction for each individual clone, the PCR reaction is carried
out on pools containing DNA extracted from many individual clones
from the library. In the same way in which it is possible to perform PCR on
a sample of human genomic DNA and to produce a single PCR product that
is specific to the set of primers used, it is also possible to perform a PCR
reaction on DNA extracted from a pool containing many individual clones.
In this case you will only amplify a PCR product if the pool from which you
made your DNA template contained the clone recognized by the primers.
You would need to perform a second round of PCR screening of each of the
individual clones in the pool to identify the clone precisely.
Various techniques have been developed to reduce the number of clones
which need to be screened. One of these is to create pools in such a way that
each clone is present in more than one sample. This effectively gives each
clone an address in the library. The principle behind this strategy is easier to