Rapid screening of a large insert BAC library for specific 16S rRNA genes using TRFLP.
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.
Babcock
D A
DA
Department of Environmental Sciences at Rutgers University, Cook College, New Brunswick, New Jersey 08901-8521, USA.
Wawrik
B
B
Paul
J H
JH
McGuinness
L
L
Kerkhof
L J
LJ
D001419:Bacteria / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics*; D015723:Gene Library; D020459:Genes, rRNA / Q000235:genetics*; D008967:Molecular Biology / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D012336:RNA, Ribosomal, 16S / Q000235:genetics*; D012623:Seawater / Q000382:microbiology
D012336:RNA, Ribosomal, 16S