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ofE. coliand yeast genomes. This cycle of clone to fingerprint to contig is amenable

to automation; however, the problem of closing the gaps between contigs remains

very difficult.

In order to define a common way for all research laboratories to order clones and

connect physical maps together an arbitrary molecular technique based on the poly-

merase chain reaction has been developed based onsequence-tagged sites(STS). This

is a small unique sequence between 200–300 bp that is amplified by PCR (Fig. 6.48).

The uniqueness of the STS is defined by the PCR primers that flank the STS. A PCR

with those primers is performed and if the PCR results in selected amplification of

target region it may be defined as a potential STS marker. In this way defining STS

markers that lie approximately 100 000 bases apart along a contig map allows the

ordering of those contigs. Thus, all groups working with clones have definable

landmarks with which to order clones produced in their libraries.

An STS that occurs in two clones will overlap and thus may be used to order the

clones in a contig. Clones containing the STS are usually detected by Southern

blotting where the clones have been immobilised on a nylon membrane. Alternatively

a library of clones may be divided into pools and and each pool PCR screened. This is

usually a more rapid method of identifying an STS within a clone and further

refinement of the PCR-based screening method allows the identification of a particu-

lar clone within a pool (Fig. 6.49). STS elements may also be generated from variable

regions of the genome to produce a polymorphic marker that may be traced through

families along with other DNA markers and located on a genetic linkage map. These

Isolate genomic cosmid clone

Subclone DNA into M13 sequencing vector

Sequence 400 to 500 bp from M13 clones

Identify unique sequence (database searching)

Design primers for PCR (20 to 25 bp sequences)

Analyse amplification products

Functional STS markers will give single product

Fig. 6.48General scheme of the production of a functional STS marker.

256 Recombinant DNA and genetic analysis