Rapid screening of a large insert BAC library for specific 16S rRNA genes using TRFLP

D. A. Babcock, B. Wawrik, J. H. Paul, L. McGuinness, L. J. Kerkhof

Research output: Contribution to journalArticlepeer-review

14 Scopus citations

Abstract

It is widely believed that the vast majority of microbes in the environment have-yet-to-be cultured using standard techniques. Bulk DNA from microbial communities is therefore often cloned into large insert vectors (e.g. bacterial artificial chromosomes [BAC] or cosmids) in order to study the genetic properties of these as yet (un)-cultured bacteria. In a typical BAC experiment, tens of thousands of clones are generated with only a small fraction of colonies containing the target(s) of interest. Efficient screening methodologies are therefore needed to allow targeted clone isolation. In this paper, we describe a rapid, inexpensive protocol that allows for the identification of specific 16S ribosomal RNA genes in a metagenomic library arrayed into 384-well microtiter plates. The rapid screening protocol employs Terminal Restriction Fragment Length Polymorphism (TRFLP) analysis to identify wells containing specific T-RF peaks. A nested approach using multiplexed samples of 384, 48, 8, and single colony analysis is described and applied in order to survey a BAC library generated from a marine microbial community off the coast of New Jersey. Screening revealed a total of 50 different 16 rRNA genes within the BAC library. Overall, the multiplexing format provided a simple, cost effective methodology for detecting clones bearing a target gene of interest in a large clone library. However, the limitations of screening BAC libraries using PCR methodologies and recommendations for improved screening efficiency using this approach are also discussed.

Original languageEnglish (US)
Pages (from-to)156-161
Number of pages6
JournalJournal of Microbiological Methods
Volume71
Issue number2
DOIs
StatePublished - Nov 2007

All Science Journal Classification (ASJC) codes

  • Microbiology
  • Molecular Biology
  • Microbiology (medical)

Keywords

  • 384-well
  • Bac library screening
  • Multiplex
  • TRFLP

Fingerprint Dive into the research topics of 'Rapid screening of a large insert BAC library for specific 16S rRNA genes using TRFLP'. Together they form a unique fingerprint.

Cite this