Date of Award
Master of Science (MS)
Dr. Larry K. Kline
The objective of this research was to survey a number of DNA samples from various organisms, using different primers, to determine what differences exist between organisms relative to each primer. This survey would help to contribute information for the following:
1. Educational purposes. A student receiving an unknown DNA should be able to identify the organism to particular taxa.
2. Determine the presence of PCR products from DNA and primers that would not typically be used together. That is, a survey of unconventional combinations.
3. Determine what differences exist between the snails, L. saxatalis and L. compressa using this particular battery of primers.
The results of this work indicate that:
1) A student would be able to narrow the choices if given one of these 12 DNAs as an unknown and this particular battery of primers. See Appendix D for an example.
2) The Universal bacterial primers amplified some eukaryotic DNA.
3) The human Alu primers amplified firefly and snail DNA as well as H. sapiens.
4) The Universal animal primers amplified V fischeri DNA and were not truly universal with the 8 DNA samples used here. H. sapiens DNA was not amplified by these primers. The DNA that was amplified gave fragments that were of variable size and not equivalent to the positive control.
5) The primers for the Histone 3 gene, one of the most highly conserved proteins, gave variable results and amplified DNA from V fischeri which does not contain histone proteins.
6) The Lux primers, specific for Vibrio fischeri amplified DNA from another bioluminescent organism, which was eukaryotic.
7) The Mitocox primers, specific for mitochondrial cytochrome C oxidase of insects, worked for both prokaryotes as well as all of the eukaryotes in the survey. The major 710 base pair fragment was present in all 12 organisms used and variable minor bands were seen in three organisms.
8) The Nrd primer, specific for the E. coli nucleotide reductase gene, proved to be specific for E. coli in this survey. This primer was used as a marker for coliform contamination such as suspected with C. elegans.
9) Additionally, L saxatalis and L. compressa do have a different fingerprint with these primers. These two snails have identical results with all the primers except Alu and Analu. L. compressa is amplified with the Alu primer while L. saxatalis is not. The Analu primer gives two bands for both DNAs, one of identical size and the other of a different length.
Kiggins, Jeffrey S., "Taxa Determination by the Polymerase Chain Reaction: a Survey" (1999). Biology Master’s Theses. 91.