Genome Walking by Single-specific-primer Polymerase Chain Reaction: SSP-PCR

We have devised a strategy to extend the use of the polymerase chain reaction (PCR) to amplify double-stranded DNA when sequence information is available only at one extremity. The only information required is a short stretch of sequence used to design a gene-specific primer, which is then used in combination with a second generic vector primer at the unknown end. The primers are used in a PCR reaction after ligating the unknown end to a generic vector. Restriction, ligation, amplification and sequencing of the products can be achieved within three days. This method eliminates the laborious steps of shotgun cloning, colony screening and culturing of cells. We have used this method to take two contiguous steps beyond the histidine transport operon in Salmonella typhimurium. We also demonstrate the usefulness of this technique to do chromosome walking in the absence of any restriction data.