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August 2006, SoftGenetics - Program Upgrades
GeneMarker® Software for Terminal-Restriction Fragment Length Polymorphism (T-RFLP) Data Analysis

Introduction

One culture-independent molecular method for fingerprinting microbial populations is terminal-restriction fragment length polymorphism (T-RFLP)(1). T-RFLP has been used to demonstrate “species diversity in activated sludge, bioreactor sludge, aquifer sand and termite guts” (2); to distinguish populations of denitrifiers, Bacteria and Archaea in marine sediments (3); to show “temporal changes in the diversity of the bacterial communities” in polluted marine environments (4); and to study fungal populations in soil (5). This sensitive high-throughput method has the potential to generate large sets of data. Improving speed and efficiency of terminal restriction fragment (T-RF) analysis will further understanding the diversity, richness and dynamics of microbial populations and its relationship to ecological processes.

T-RFLP is a polymerase chain reaction (PCR) based genetic fingerprinting technique. DNA is extracted from a microbial community. One of the primers of a primer pair is labeled with a fluorescent dye and used to amplify a selected region of a gene of interest by PCR. The resulting PCR fragment is digested with one (sometimes two or more) restriction endonuclease and the T-RFs are separated with an automated DNA analyser. Microbial diversity in a community can be estimated by analysing the number and peak heights of T-RF patterns (2).

GeneMarker® can perform fragment analysis and genotyping on four or five colour data sets from any slab gel or capillary electrophoresis system. This software automatically corrects for many common problems - instrument spike, colour pull-up, peak pull-up, noisy data, saturated peaks and stutter peaks - saving significant analysis time and cost, efficiently analysing raw fragment data within seconds. GeneMarker® is robust software to analyse DNA fragment data labeled with MegaBACE™ dyes (Amersham), Big Dye® (AppliedBiosystems Inc.) or Beckman dyes from a variety of platforms: ABI DNA Analyser or Genetic Analyser, Amersham instruments or Beckman instruments. GeneMarker is compatible with files from all major capillary and slab gel electrophoresis systems including ABI files (*.FSA, *AB1, *.ABI), SCF files, MegaBace® files (*.RSD, *.ESD), SpectruMedix files (*.SMD, *.SMR), Beckman files and Li-Cor files. The software features high efficiency allele calling, adjustable parameters and a variety of reporting options.

 For more details, download the T-RFLP PDF PDF icon (198Kb)

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