PCR and DNA Sequencing Experiment

PCR and DNA Sequencing Experiment

PCR and DNA Sequencing

PCR and DNA sequencing were used to identify a bacterium isolated from a patient. The bacterium was determined to be Bartonella henselae. B. henselae is associated with cat scratch disease (CSD) and is transmitted to humans via cats.

Polymerase chain reaction (PCR) has been used to amplify segments of DNA for many years. It is a very useful tool for further molecular manipulations with a piece of DNA (Liang). Also, it makes the study of bacteria that are difficult to grow in laboratory conditions easier since only the DNA is needed for PCR not pure colonies. PCR takes advantage of a highly conserved strand of bacterial DNA that can be used as universal primers to make copies of bacterial DNA called 16S rDNA. Now, using these conserved 16S rDNA primers and fluorescently labeled terminal nucleotides in a PCR one can make a lot of small fragments of DNA copies that then can be used to sequence the DNA strand (Hiraishi). The resulting fragments are then separated electrophoretically in a DNA sequencing machine that also reads the fluorescent labels of the terminal nucleotides thus determining the sequence of the small overlapping fragments, which are then used to construct the entire DNA sequence. The purpose of this research was to identify a bacterium from a patient sample using PCR and DNA sequencing methods. The bacterium was identified to be Bartonella henselae.

Materials and Methods

A patient sample was taken and isolated bacterial colonies were grown from this sample on solid media. Bacterial DNA was extracted from an isolated colony by breaking down the cell wall with proteases and separating the cell debris from the DNA by centrifuging. The supernatant containing the bacterial DNA was then amplified using standard PCR methods (Mullis). An initial incubation at 95°C for ten minutes was followed by 30 cycles of the following conditions: Melt at 95°C for 30 seconds, anneal at 60°C for 30 seconds, and extend at 72°C for 45 seconds. The PCR is completed by a final extension at 72°C for 10 minutes then stored at 4°C. Next, the amplified bacterial DNA was purified using a microconcentrator column. A buffer and the DNA sample were applied to the column and centrifuged at 3000 rpm for fifteen minutes, which collects the amplified DNA on the column. The column is then inverted into a clean tube and washed with another buffer via centrifugation at 3000 rpm for two minutes to release the DNA from the column into the collection tube. The purified DNA is then prepared using a PCR like procedure that is described in detail by Innis and then can be automatically sequenced using standard methods (Hirashi). The resulting DNA sequence can then be entered into the NCBI database to search for a bacterial match. The database can be found at the following web address: http://blast.ncbi.nlm.nih.gov/Blast.cgi.

Results

Using PCR and DNA sequencing techniques it was determined that the bacteria isolated from a patient sample was Bartonella henselae. The results of the DNA sequencing can be seen in figure one. This is the output that was inputted into the NCBI database to search for a bacterial match. The top five results of the NCBI database search for the DNA sequence can be seen in table one. The results indicate that the bacterium was likely Bartonella henselae. Rochalimaea is an old genus name for Bartonella.