Gene Cloning

Further Routes to Gene Identification 161
6.7 Identification of a Linked Marker
Although we have been talking up to now in terms of beginning a positional
cloning project by finding a known gene which is linked to the gene of
interest, in practice the known gene does not have to be a gene in the classical
sense of the word; that is, a stretch of DNA which codes for a protein.
Cloning experiments in fact rely far more on polymorphisms which are not
in genes at all, but rather are found in the non-coding DNA which makes
up the bulk of the human genome (Section 2.3). Initially, the most widely
used were restriction fragment length polymorphisms (RFLPs) (Box 6.3).
RFLPs are not genes, but simply points in the DNA where there are
sequence differences between individuals. However, they are of course
inherited exactly like traditional genes which code for proteins, and hence
can be used in linkage analysis to build up detailed maps of the genome.
What RFLPs do is to give us a series of markers which are specific for particular
positions on the genome. When we wish to clone a particular gene
for which we have no information, one of the first steps we can therefore
take is to try to identify RFLPs which are close to this gene and are hence
tightly linked to it. The more tightly linked a given RFLP is with a particular
phenotype the more useful it is, since we can now attempt to move from
this RFLP to the gene we are after, as discussed in the next section. The
principle of linking an RFLP to a particular gene of interest is shown in very
simplified form in Figure 6.10.
RFLPs have now been largely superseded in positional cloning by other
polymorphisms which are more frequent and more informative. The most
useful of these are SNPs or single nucleotide polymorphisms (see Section
2.3). However, the broad principles of how these polymorphisms can be
used in these sorts of experiments are the same, and these other polymorphisms
will not be discussed further.
6.8 Moving From the Marker Towards the Gene of Interest
Finding a marker linked to the gene that we want is not the same as finding
the gene. Indeed, even for very tightly linked markers, the actual physical
distance from the marker to the gene of interest may still be many thousands
of bases. Having identified a polymorphism that is close to our gene
of interest, where do we go next? The aim of subsequent experiments is to
construct a detailed map of the region adjacent to the RFLP, and to clone
the DNA corresponding to the adjacent region, both of which will assist in
finding the gene of interest. To do this, a number of methods are used,
among them chromosome walking and chromosome jumping. In chromosome
walking, an initial cloned fragment, shown by mapping to be linked
to the gene of interest, is hybridized against a genomic library. Clones that
hybridize to the initial cloned fragment will represent stretches of DNA
which are adjacent to the initial fragment. By taking these clones, and rescreening
the genomic library with them, the next fragment along can be