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Direct sequencing of single primer PCR products: a rapid method to achieve short chromosomal walks.

One of the limitations of PCR to analyse a region of interest is that it requires knowledge of the flanking sequences in order to design amplification primers. Several adaptations of PCR have been described which allow some unknown sequences adjacent to regions of known sequence to be amplified. Primers directed to repetitive sequences, for instance Alu repeats can be used to pair with a primer specific to the region of interest (1), frequently however the nearest repeat lies too far away to allow PCR to proceed. In another approach known as inverse PCR the target DNA is circularised before amplification, allowing extension to proceed outwards from the region of interest (2). We have frequently observed a PCR artefact where a single primer generates products when used in a PCR reaction alone. This artefact has been previously reported and used to clone complementary sequences from genomic DNA (3). By combining this technique with direct sequencing of pooled PCR products we have improved the yield of the PCR reactions and by cutting out the cloning steps we have greatly speeded up the method. We have used this technique to make short chromosomal walks in the Human CD44 locus from a yeast artificial chromosome (YAQ (see below), and also in our laboratory to obtain the promoter sequence proceeding the TCRBV2S1 gene in the human T-cell receptor locus from genomic DNA. This technique will have useful applications in sequencing DNA flanking regions of known sequence such as the ends of yeast artificial chromosomes, DNA flanking the insertion of transgenes or retroviruses, and sequencing across intron-exon boundaries and promoters from genomic DNA.