RFLP analysis—DNA sequence polymorphisms display different migration profiles from wild-type fragment patterns when DNA is digested with restriction fragments and separated by size using electrophoresis.

T-RFLP (terminal-RFLP) analysis—T-RFLP is a culture-independent RFLP method (only terminal fragments are labeled and detected) used to study high-complexity microbial populations based on variations in 16S rRNA (ribosomal RNA). This technique allows you to examine complex communities without the need for any genomic sequence information. This method is rapid, sensitive, reproducible, and enables you to study bacterial populations in diverse natural habitats and to analyze the organisms’ responses to changes in environmental or physiological parameters.

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Figure 1: Flowchart of steps required for a T-RFLP assay.

Typical T-RFLP analysis comprises five major steps (Figure 1, click to enlarge.)

  1. DNA isolation and purification
  2. PCR amplification and restriction enzyme digestion
  3. Separation and detection of the digested products via electrophoresis
  4. Analysis of data to generate the fragment profile for each sample
  5. Clustering analysis based on the profile of samples from step 4

Step-by-Step Guide to RFLP Analysis

DNA Purification kit

DNA extraction is a critical first step in the experimental workflow of DNA Sequencing and Fragment analysis. The overall quality, accuracy and length of the DNA sequence read can be significantly affected by characteristics of the sample itself, and the method chosen for nucleic acid extraction. Ideal methods will vary depending on the source or tissue type, how it was obtained from its source, and how the sample was handled or stored prior to extraction.


Recommended Products: DNA Isolation

PCR Kit

PCR of 16S rRNA fragment with 5'-fluorescent labeled primer pair.

Learn MoreRecommended Products

Digest PCR amplicons with restriction enzyme(s).

Recommended Products: Perform Restriction Digestion

Which Enzyme to Choose?

Fragment Analysis sizing graph image.

Add aliquot of PCR product to size standard and Hi-Di Formamide mix; heat denature and proceed with electrophoresis.

Recommended Products: Prepare Sample for Analysis

Sizing Information

To learn more about our GeneScan Size standards used in sizing experiments please see our table below.

When DNA fragments are labeled with: Choose a size standards labeled with: Recommended Size Standard Possible Applications Other Kits and Products
dR110, dR6G, dTAMRA™, dROX (Dye Set DS-02 - Filter E5)LIZ®
GeneScan™ 120 LIZ® Size Standard
GeneScan™ 600 LIZ Size Standard v2.0
SNaPshot®
Microsatellite, LOH, ISSR, Chimerism, ISSR, RFLP, T-RFLP, MSMSA
SNaPshot® primer focus kits
Custom Labeled Primer
5-FAM™, HEX™, NED™ (Dye Set DS-30 - Filter D)ROX™GeneScan™ 500 ROX™ Size StandardCustom Fragment AnalysisCustom Labeled Primer
6-FAM™, VIC®, NED™ (Dye Set DS-31 - Filter D**)ROX™GeneScan™ 500 ROX™ Size Standard

Custom Fragment Analysis

Custom Labeled Primer
5-FAM™, JOE, NED™ (Dye Set DS-32 - Filter F)ROX™GeneScan™ 500 ROX™ Size Standard
Microsatellite (Forensic)
AFLP
AmpFℓSTR® Kits,
Plant/Microbial AFLP Kits®
6-FAM™, VIC®, NED™, PET® (Dye Set DS-33 - Filter G5)LIZ®GeneScan™ 600 LIZ Size Standard v2.0
Microsatellite (Forensic)
Pathogen Detection
Custom Fragment Analysis
AmpFℓSTR® Kits,
TrueScience™ RespiFinder®
Custom Labeled Primer
Capillary Electrophoresis

During capillary electrophoresis, the products of the PCR are injected electrokinetically into capillaries filled with polymer. High voltage is applied so that the fluorescent DNA fragments are separated by size and are detected by a laser/camera system.

Which Electrophoresis Instrument (Genetic Analyzer) Is Right for You?

  Instrument
 3730xl
3730xL
3730
3730
3500xl
3500xL
3500
3500
3130xl
3130xL
3130
3130
310
310
Number of Capillaries96482481641
Compatible Applications:
(S) Supported; (A) AB Demonstrated; (C) Customer Demonstrated; (N) Not Supported
Conformation Analysis
- SSCPNNNNAAS

Analyze the RFLP profile.

Which Data Analysis Software Is Right for You?

Highlighted products for RFLP Analysis:

Verti Thermal Cycler

3500 Series Genetic Analyzers

GeneMapper® Software