Restriction Fragment Length Polymorphisms - RFLP

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