Nonspecific, nested suppression PCR method for isolation of unknown flanking DNA

Richard Tamme, Esther Camp, R. Daniel Kortschak, Michael Lardelli

Research output: Contribution to journalArticlepeer-review

10 Citations (Scopus)

Abstract

We report the development of a simple, sensitive and robust two-step PCR method for the isolation of unknown sequences flanking characterized regions of genomic DNA or cDNA. The method requires 100 bp or less of a known sequence upstream of an oligonucleotide primer binding site. A first round of suppression PCR is conducted at low stringency with a polymerase lacking exonuclease activity to generate a mixture of products including fragments of the desired flanking sequence that are often greater than 1 kb in length. The desired fragments are then amplified from the mixture in a second round of suppression PCR using an extended oligonucleotide in combination with a polymerase exhibiting exonuclease activity. These fragments are subsequently identified by hybridization with 100 bp of known sequence or simply by cloning sequencing. The method is widely applicable and allows isolation of novel cDNA from very low abundance transcripts.

Original languageEnglish
Pages (from-to)895-902
Number of pages8
JournalBioTechniques
Volume28
Issue number5
DOIs
Publication statusPublished or Issued - 2000
Externally publishedYes

ASJC Scopus subject areas

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

Cite this