Terminal restriction fragment length polymorphism monitoring of genes amplified directly from bacterial communities in soils and sediments.

Abstract:

:Terminal Restriction Fragment Length Polymorphism (T-RFLP) or Fluorescent Polymerase Chain Reaction/Restriction Fragment Length Polymorphism (FluRFLP) have made a significant impact on the way in which PCR products amplified from mixed community DNA extracts have been assessed. Technically, these approaches are essentially the same. PCR products are generated that contain at one 5' end label, typically a fluorescent moiety, that will be detected by a DNA sequencing machine. Upon digestion using a specific restriction endonuclease, labeled and unlabeled fragments are generated. This restriction endonuclease is chosen such that following this digestion, each labeled fragment corresponds to a different sequence variant. During electrophoretic separation, the DNA sequencing machine detects only these labeled fragments and therefore detects only the sequence variants. The aim of this article is to describe the protocols and demonstrate that this profiling can be performed using different DNA sequencing machines. The analysis and applications of this approach are also discussed.

journal_name

Mol Biotechnol

journal_title

Molecular biotechnology

authors

Bruce KD,Hughes MR

doi

10.1385/MB:16:3:261

keywords:

subject

Has Abstract

pub_date

2000-11-01 00:00:00

pages

261-9

issue

3

eissn

1073-6085

issn

1559-0305

pii

MB:16:3:261

journal_volume

16

pub_type

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