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.
Figure 1: Flowchart of steps required for a T-RFLP assay
Typical T-RFLP analysis comprises five major steps (Figure 1, click to enlarge.)
DNA isolation and purification
PCR amplification and restriction enzyme digestion
Separation and detection of the digested products via electrophoresis
Analysis of data to generate the fragment profile for each sample
Clustering analysis based on the profile of samples from step 4
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.
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?