In this lab, you will isolate, amplify, and sequence a piece of DNA from the mitochondria of your own cheek cells. The lab will introduce you to several vital biological concepts and techniques:
• Mitochondrial DNA. The fact that mitochondrial DNA have their own DNA is powerful evidence that they originated from the engulfment of prokaryotic cells by other cells. A question to think about: in this lab, how will you distinguish between your mitochondrial DNA and your chromosomal DNA?
• Polymerase Chain Reaction (PCR). This technique earned inventor Kary Mullis a Nobel Prize in 1993. In essence, it allows trace amounts of DNA to be easily and quickly amplified. PCR has thus provided a huge boost to areas such as DNA forensics, pharmaceutical research, and genome sequencing. The technique uses DNA polymerase isolated from Thermus aquaticus, a bacterium that lives at high temperatures. Why do you think is it important to use DNA polymerase from a thermophilic organism?
• Running a gel. This is a very common technique for checking sizes of DNA fragments. DNA, being negatively charged, migrates toward the positive electrode, with smaller fragments migrating faster. The lengths of unknown pieces are estimated via comparison to standards of known length. In your case, you will run a gel to confirm that you got a PCR product of about 400 base pairs.
• DNA sequencing. The basic strategy of modern sequencing techniques was invented by Frederick Sanger, who won a Nobel Prize for this work in 1980. We will learn more about this work in class on October 26th. For now, simply note that DNA sequencing is an essential component of most modern biology research. Once you have the sequence of a gene, you can determine its amino acid sequence, assess how closely related it is to genes from other organisms, guess its function based on comparisons with other well-characterized genes, examine how its expression is regulated, and put copies of it into other organisms, among other possibilities. A question to think about: how do the primers used for DNA sequencing compare with the primers used for PCR?
Mitochondrial DNA (mtDNA)
The high sensitivity of mtDNA analysis allows forensic scientists to obtain information from items of evidence associated with homicides or other criminal investigations, body identifications, cold cases, and small pieces of evidence containing little biological material. The maternal inheritance of mtDNA allows scientists to compare the mtDNA profile from the evidence (hairs, bones, etc.) to that of reference samples from the individual; the individuals mother, brother(s), sister(s); or any other maternally related family member. These samples should have the same mtDNA profiles because all maternal relatives inherit the same mtDNA. Since mtDNA is maternally inherited and multiple individuals can have the same mtDNA type, unique identifications are not possible using mtDNA analyses. However, mtDNA is an excellent technique to use for obtaining information in cases where nuclear DNA analysis is not feasible.
The FBI Laboratory began conducting studies on the feasibility of mitochondrial DNA (mtDNA) analysis for human identity testing in the late
1980s. Laboratory research began on a protocol for using mtDNA sequencing in forensic casework in 1992. After the sequencing technique was validated, examinations on evidentiary samples began in June 1996.
MtDNA sequencing is often used in cases w here biological evidence may be degraded or small in quantity. Cases in which hairs, bones, or teeth are the only evidence retrieved from a crime scene are particularly well
suited to mtDNA analysis. Missing persons cases can benefit from mtDNA testing whe n skeletonized remains are recovered and compared to samples from the maternal relatives or personal effects of missing individuals. Also, hairs recovered at