Random amplified polymorphic DNA PCR (RAPD-PCR) is an effective technique for studies of population and conservation genetics, taxonomy, genetic variability and insights into geographic origins and invasion routes of colonizing species. The principle behind RAPD-PCR is the detection of polymorphisms through the PCR amplification of DNA fragments with primers of arbitrary nucleotide sequence. Polymorphisms are detected as either the presence or absence of bands, caused by nucleotide sequence differences between individuals.
Figure 1-Reproducibility of RAPD-PCR markers. From left to right, duplicate RAPD patterns obtained with primers RAPD 1, RAPD 2, RAPD 3, RAPD 4 and RAPD 5 with P. latipinna genomic DNA.
Figure 2-RAPD banding profile of primers RAPD 1, RAPD 2, RAPD 3, RAPD 4 and RAPD 5. From left to right, duplicates of noble crayfish Astacus astacus (C1), freckled crayfish Cambarus maculates (C2), goldfish Carassius auratus (F1) and molly Poecilia latipinna (F2). Arrows indicate bands shared by both C. auratus and P. latipinna.
The ten decamer oligonucleotide primers used were arbitrarily designed with 50 and 60% G + C content. These primers were tested in PCR amplification on C. auratus and A. astacus DNA for their capability of producing clear bands. Five primers were subsequently chosen from these ten for use with all four species. Reproducibility of RAPD-PCR results was successfully achieved (Figure 1) by holding variables known to yield divergent results constant, such as DNA concentration. The five primers generated 90 bands for the crayfish DNA, 100% of which were polymorphic. The five primers generated 131 bands for the fish DNA, 94.7% of which were polymorphic. A high level of polymorphism was observed at the interspecific level,suggesting a high degree of genetic variability between the pairs of species compared. For future studies, the use of more primers would likely reveal a greater degree of similarity between the species evaluated and decrease the level of polymorphism observed. Also, the incorporation of additional species into the analysis would allow degrees of relatedness to be determined.
I would like to thank Jinsong Qiu and Lisa Sarran for their continuous help and enduring patience. I would also like to thank Dr. Berish Rubin for his helpful assistance and for the use of his laboratory. In addition, I thank Dr. James Fetzner at the Carnegie Museum of Natural History in Pittsburgh for his generous contribution of crayfish genomic DNA. Last but not least, I thank my classmates for providing good cheer and encouragement.
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