Biology Reference
In-Depth Information
v) Terminal restriction fragment length polymorphism (T-RFLP): The fi fth method is just an
extension of the second method described by Liu et al . (1997) where the initial steps of DNA isolation,
PCR amplifi cation and digestion with restriction enzymes are similar to those used for ARDRA.
However, in this method one of the primers used is labelled at 5'-terminus with a fl uorescent dye
and the terminal restriction fragments (T-RFs) are separated by electrophoresis and their fl uorescence
measured. T-RFLP analysis provides quantitative data about each T-RF detected, including size in base
pairs and intensity of fl uorescence (represented by peak height). Due to terminal RFLPs (T-RFLPs)
found in 16S rDNAs, this method can be used with DNA from complex microbial communities. It is
important to select proper PCR primers as well as restriction enzymes (Fig. 13). Computer simulated
analysis of T-RFLP for 1,002 eubacterial sequences, 686 sequences could be PCR-amplifi ed and
Figure 13: Flow-chart depicting different steps in T-RFLP technique.
Search WWH ::




Custom Search