DNA Hybridization Studies Of Borrelia Using Isolated Plasmid DNA To Probe Southern Blots Of Undigested Whole DNA And PST 1 Digested Whole DNA

Abstract

Borrelia spirochetes, the etiologic agents of Lyme disease, are unique among bacteria because they contain linear, rather than circular, molecules of DNA. In addition, a significant amount, sometimes up to 25%, of the bacteria’s genome, is in the form of plasmids, small pieces of DNA which can replicate autonomously and can contain functional genes. This study was undertaken to investigate the structure and origin of several unusually large plasmids in four isolates of Borrelia, HO14, BO23, VS116, and R100. Isolating the desired plasmids from the bacterial cells was accomplished through gel electrophoresis, making it possible to isolate the desired plasmid by extracting it from the gel. After the plasmid-containing band was cut out of the gel, the DNA was purified and cut with restriction endonucleases. The DNA was then labeled with radioactive phosphorus and used to probe fractionated DNA previously transferred to a membrane. After allowing the radioactive probe to hybridize to the membrane-bound DNA, the membrane was covered with Hyperfilm. The radioactive molecules caused the film to be exposed, making it possible to determine which bands hybridized with the probe. In this project, I tried to determine the origin of the large plasmids in question. Were they part of the bacterial chromosome that had somehow become excised? Or were they created from a smaller plasmid that had made a mistake during replication and formed a dimer?