The Polymerase Chain Reaction-Restriction Fragment Length Polymorphism Technique - Essay Example

The use of the technique like PCR-RFLP gives a good correlation between classical RFLP data and sequence-based phylogenetic distance analysis. It also validates the use of mitochondrion DNA as a genetic marker for parental identification and for identification of hybridization. In the conclusion, the author demonstrates how different methods of phylogenetic analysis give different results? Thus, it is very important to draw the right conclusion considering the limitations of each technique.

                  The paper starts with the hypothesis demonstrating mitochondrial DNA as a primary tool for investigation of evolutionary diversion among closely related species and the development of a new approach in terms of PCR-RFLP-based rapid and inexpensive techniques to established phylogenetic correlation among different species. They also investigated the correlation between two different approaches and indicated that data obtained by two different techniques may not be identical and hence caution must be taken to interpret them. For validation of the hypothesis, the authors selected four avian sp. found in North America, those having a high rate of hybridization namely, Dendroica occidentalis, D. Townsend, D. virens, and D.nigrescens. To investigate above mention hypothesis the first experiment was based on the classical RFLP-based technique. The total mitochondrial DNA was Isolated and digested with 14 restriction enzymes to obtained a band pattern which was subsequently analyzed by David L. Swofford’s pup* 4.0d64 program for calculation of Nei–Li distances. Similarly, for sequences-based analysis, three genes located on two sites on mtDNA were selected and sequenced, namely 681bp cytochrome oxidase I, and 1074bp ATP synthase8, and 6 genes from 30 representative warbler individuals.  All these sequences were used to calculate K2 and MP analysis. For the development of PCR-RFLP-based techniques, mt DNA of two species was scanned in regions where COI and ATPase were located for identification of sites having nucleotide sequence variations. Amongst the various sites few restriction sites were selected, those which are recognized by commercially available restriction enzymes. Out of six diagnostic sites, on COI, two sites were selected having restriction sites for XmnI. MspI and AluI. Based on sequence information, primers were designed to amplify those regions, and after amplification PCR product was used for RFLP analysis using 4 restriction enzymes.

                                  Comparison of sequence data with RFLP data shows good correlation when autocorrelation function was applied but once autocorrelation function was removed some differences among species origin were observed. The chronological clock gave a different timeline for the origin of species in the case of RFLP and sequence-based data. For evaluating the use of the PCR-RFLP system they employed two populations of Townsend and occidentalis. Ancestral townsendi were represented by 39 individuals from eastern British Columbia, north-central Alaska, and eastern Oregon. Ancestral occidentalis was represented by 19 individuals from southern Oregon and northwestern California.