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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.
Discussion
PCR and DNA sequencing was successfully used to identify a bacterium that was isolated from a patient sample. The bacterium was identified to be Bartonella henselae. B. henselae is known to cause cat scratch disease and is transmitted to humans via a cat vector. Some common symptoms of cat scratch disease are swelling at the site of injury, fever and fatigue. It is generally not treated but in more severe cases antibiotics may be prescribed. In the future, the experiment could be repeated for other isolated colonies grown from the patient...
"Direct automated sequencing of 16S rDNA amplified by polymerase chain reaction from bacterial cultures without DNA purification." Applied Microbiology15(5) (2008): 210-213. Web. 11 Aug. 2010.
Innis, M.A., Myambo, K.B., Gelfand, DH, Brow, M.D. "DNA sequencing with Thermus aquaticus DNA polymerase and direct sequencing of polymerase chain reaction-amplified DNA." Proceedings of the National Academy of Sciences, USA 85 (1988): 9436-9440. Web. 11 Aug. 2010.
Liang, Q., Chen, L., Fulco, A.J.. "An efficient and optimized PCR method with high fidelity for site-directed mutagenesis." PCR Methods and Applications 4(5) (1995): 269-274. Web. 11 Aug. 2010.
Mullis, K.B.. Target amplification for DNA analysis by the polymerase chain reaction. Annals of Clinical Biology (Paris) 48(8) (1990): 579-582. Web. 11 Aug. 2010.
Figure
1
Accession Number
Description
gi|39295|Z11684.1
R.henselae 16S rRNA gene gi|6626180|AF214556.1
Bartonella henselae 16S ribosomal RNA, partial sequence gi|2828303|AJ223779.1
Bartonella henselae 16S rRNA gene, isolate FR96/BK38
gi|2828302|AJ223778.1
Bartonella henselae 16S rRNA gene, isolate FR96/BK3
gi|109501457|DQ645426.1
Bartonella henselae strain M40SHD 16S ribosomal RNA gene, partial sequence
References
Hiraishi, A. "Direct automated sequencing of 16S rDNA amplified by polymerase chain reaction from bacterial cultures without DNA purification." Applied Microbiology15(5) (2008): 210-213. Web. 11 Aug. 2010.
Innis, M.A., Myambo, K.B., Gelfand, DH, Brow, M.D. "DNA sequencing with Thermus aquaticus DNA polymerase and direct sequencing of polymerase chain reaction-amplified DNA." Proceedings of the National Academy of Sciences, USA 85 (1988): 9436-9440. Web. 11 Aug. 2010.
Liang, Q., Chen, L., Fulco, A.J.. "An efficient and optimized PCR method with high fidelity for site-directed mutagenesis." PCR Methods and Applications 4(5) (1995): 269-274. Web. 11 Aug. 2010.
Mullis, K.B.. Target amplification for DNA analysis by the polymerase chain reaction. Annals of Clinical Biology (Paris) 48(8) (1990): 579-582. Web. 11 Aug. 2010.
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