Retrieval of entire genes from environmental DNA by inverse PCR with pre-amplification of target genes using primers containing locked nucleic acids

Kazutaka Yamada, Takeshi Terahara, Shinya Kurata, Toyokazu Yokomaku, Satoshi Tsuneda, Shigeaki Harayama

Research output: Contribution to journalArticle

29 Citations (Scopus)

Abstract

We had been unsuccessful to amplify desired nucleotide sequences from various environmental DNA samples by using the inverse polymerase chain reaction (IPCR) technique, most probably because the copy numbers of target DNA sequences had been quite low. To enrich the target DNA sequences prior to IPCR, a rolling-circle amplification was used with a site-specific primer containing locked nucleic acids (LNAs). This pre-amplified IPCR (PAI-PCR) method increased the sensitivity of PCR almost 10 000 times compared with the standard IPCR in model experiments using Escherichia coli. We then applied the PAI-PCR method to isolate glycosyl hydrolase genes from DNAs extracted from vermiform appendixes of horses and termite guts. The flanking sequences of the target genes were amplified and cloned successfully using PAI-PCR, whereas standard IPCR resulted in no amplification.

Original languageEnglish
Pages (from-to)978-987
Number of pages10
JournalEnvironmental Microbiology
Volume10
Issue number4
DOIs
Publication statusPublished - 2008 Apr

ASJC Scopus subject areas

  • Microbiology
  • Ecology, Evolution, Behavior and Systematics

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