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Journal of Undergraduate Research

Keywords

mitochondrial DNA mutation, myopathies, lactic acidosis, mtDNA

College

Life Sciences

Department

Microbiology and Molecular Biology

Abstract

This research focused on the search for the probable mitochondrial DNA (mtDNA) mutation in one family affected with fibromyalgia and lactic acidosis. The hypothesis was that there is mixture of normal and mutated mtDNA, called heteroplasmy. I first obtained the four patients’ consent to participate in the study. The four patients were between the age of 19 and 25 years-­‐two males and two females. They donated urine samples to be used for DNA analysis. I isolated DNA using a Qiagen DNA extraction kit and obtained concentration measurements of 8-­‐ 10 nanograms per microliter using the nanodrop spectrophotometer in the RIC facility. I used the Taq polymerase reaction mixture for PCR amplification of the mtDNA using 17 sets of primers. These 17 segments of overlapped to provide coverage of the whole circular mitochondrial genome. I ran these reaction samples on DNA gel electrophoresis for about an hour to check that I had plenty of amplified DNA. I used the restriction enzyme, Surveyor Nuclease, to check for any mismatched mutant/nonmutant DNA that could have formed during the PCR reaction. I was so excited when I saw that one of the segments was cut by the restriction enzyme. I thought I had found the mutation location in the cytochrome b gene.

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