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polymorphic STSs are useful since they may serve as markers on both a physical map

and a genetic linkage map for each chromosome and therefore provide a useful

marker for aligning the two types of map.

6.9.2 Gene discovery and localisation

A number of disease loci have been identified and located to certain chromosomes.

This has been facilitated by the use ofin situmapping techniques such as FISH. In fact

a number of genes have been identified and the protein determined where little was

initially known about the gene except for its location. This method of gene discovery

is known as positional cloning and was instrumental in the isolation of theCFTRgene

responsible for the disorder cystic fibrosis (Fig. 6.50).

The genes that are actively expressed in a cell at any one time are estimated to be

as little as 10% of the total. The remaining DNA is packaged and serves an as yet

unknown function. Investigations have found that certain active genes may be

identified by the presence of so-calledHTF(HpaII tiny fragments) islands often found

at the 5^0 end of genes. These are CpG-rich sequences that are not methylated and

form tiny fragments on digestion with the restriction enzymeHpaII. A further gene

discovery method that has been used extensively in the past few years is a PCR-based

technique giving rise to a product termed anexpressed sequence tag(EST). This

represents part of a putative gene for which a function has yet to be assigned. It is

carried out on cDNA by using primers that bind to an anchor sequence such as a poly(A)

tail and primers which bind to sequences at the 5^0 end of the gene. Such PCRs may

Genomic YAC clone (150 kb) Overlapping cosmid contigs

Physical map (5 to 10 kb)

STS marker

200 to 300 bp PCR-STS

Fig. 6.49The derivation of an STS marker. An STS is a small unique sequence of between 200 and 300 bp that

is amplified by PCR and allows ordering along a contig map. Such sequences are definable landmarks with

which to order clones produced in genome libraries and usually lie approximately 100 000 bp apart.

257 6.9 Analysing whole genomes