Rapid acquisition of unknown DNA sequence adjacent to a known segment by multiplex restriction site PCR

Kristy L. Weber, Mark E. Bolander, Gobinda Sarkar

Research output: Contribution to journalArticle

Abstract

The determination of unknown DNA sequences around a known locus has important applications in molecular genetics, specifically in genomic walking and genome mapping. Several PCR-based methods have been reported to address this issue, but they often involve multiple, time-consuming steps. We have previously described a technique known as restriction site PCR (RS-PCR) that allows sequence acquisition faster than the existing methods. The method involves PCR using four separate universal primers that are representative of given restriction enzyme sites (RS primers), and a specific primer from one end of the known sequence. We have now significantly improved the technique by mixing the four universal primers into one PCR tube with the first specific primer. This is followed by a nested PCR with the mixed RS primers and an internal specific primer, after which the product is sequenced by direct automated sequencing. The technique, called multiplex RS-PCR (mRS- PCR), is reproducible and can be used to obtain unknown sequence adjacent to known sequences in both the upstream and downstream directions. We illustrate the application of mRS-PCR in the acquisition of approximately 780 bp of genomic sequence starting from a known sequence of approximately 120 bp. Multiplex RS-PCR appears to be the fastest of all methods that address the issue of unknown sequence retrieval adjacent to a known region.

Original languageEnglish (US)
Pages (from-to)415-419
Number of pages5
JournalBioTechniques
Volume25
Issue number3
DOIs
StatePublished - 1998

ASJC Scopus subject areas

  • Biotechnology
  • Biochemistry, Genetics and Molecular Biology(all)

Fingerprint Dive into the research topics of 'Rapid acquisition of unknown DNA sequence adjacent to a known segment by multiplex restriction site PCR'. Together they form a unique fingerprint.

  • Cite this