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 
(198Kb)
