PCR and DNA Sequencing of DNA Gap in Arhodomonas sp. Seminole

Abstract

Arhodomonas Seminole is an aerobic, halophilic bacterium enriched from salty, crude-oil-impacted soil and has been discovered in Seminole Co, Oklahoma. About 8-10% of the genes within the genetic sequence of Arhotomonas Seminole are broken between two pieces, totaling about 750 separate contigs. Our goal was to generate DNA sequence data for a gap in a particular region in order to bring 2 larger pieces together. We needed to predict adjacent contigs based on what is known in a related species, and we were going to prove this using processes such as PCR and DNA sequencing. In order to complete the PCR reaction, we required forward and reverse primers. We obtained these by using fused contig sequences that we inputted into a blastX website, which then outputted the primer sequences. Once we had these primers we were able to complete our PCR reaction and proceed to a process called Agarose gel electrophoresis. This process allowed us to visualize our results and determine the DNA fingerprint, which was revealed in the pattern of the PCR product size. This allowed us to determine that the protein that connected our upstream and downstream contigs was Thioalkalivibrio sp. ALJ17, which confirmed that the two contigs were in fact adjacent.