The mitochondrial (mt) genome which contains only 37 genes, is used for the process of the production of energy and its storage in ATP. In the "Human Mitochondrial DNA Kit" background, it said that there was strong evidence that mitchondria once existed as a free living bacteria, which were taken up by primitive ancestors of eukaryotic cells. The MT genome has some bacteria like feature, is also a circular molecule and very few noncoding sequences (introns) interrupt mt gene. In 1981, the entire DNA sequence of the mt genome was determined. In the mt genome, there is a region of noncoding nucleotides (1,200) in which it contains signals that control replication of the chromosome and the transcription. So this DNA sequence is called the hypervariable because it accumulates mutations at approximately 10 times the rate of nuclear DNA, and because of this, ther is a unique pattern of single nucleotide polymorphyism, which could be inherited through generations. Also in the 1980's, Alan Wilson of UC Berkeley, used mtDNA polymorphisms to create a family tree, that showed the ancestral relationships of modern people. From this they were able to conclude that all modern humans arose from Africa about 200,000 years ago. Today mt DNA has been used to identify the remains of unkown soldiers in Vietnam, also determine the reamins of the Romanov royal family and the relationship of Neandertal remains to modern humans
Directions:
Step 1. First we labeled one screw cap tube containing 200 hl of InstaGene matrix and then rinsed our mouth with saline solution for 30 secounds. (chewed inside of our gums to loosen cheek cells.).
Step 2. Then we placed this saline solution that we spit out into the cap tube containing the InstaGene and then we centrifuged it for about 2 minutes.(After it was centrifuged, there was a white bead of whitish cells at the bottom of the tube.)
Step 3. Then after centrifuging, we poured out the extra saline solution out of the tube, so then we only had the pellet of white cells at the bottom of the tube.
Step 4. Then we vortexed the tube so there would be no clumps of cells remaining and to resuspend the pellet of cells.
Step 5. Then we transfered the resuspended cells, into the screwcap tube containing InstaGene.
Step 6. Then we vortexed the tube and then placed the tube into a water bath (56 degrees Celcius) for 10 minutes.
Step 7. After we placed the tube in the vortex again and then put it into a boiling water bath (100 degrees Celcius) for 5 minutes.
Step 8. Then the next day we placed the mixture into a PCR tube and also added some yellow master mix. Then we placed it into the thermal cycler for 40 cycles.
Step 8. Finally, we added loading dye to our PCR tubes and then placed our mixtures into the wells of the agrose gel and then turned on the electrophoresis apparatus for about 10 minutes.
Results: Our results showed that we were all negative for the disease, but it could possibly be wrong because we could have messed up when loading the mixtures into the wells of the agrose gel. Since our results were very faint and very hard to see if they really were negative for the disease. I also think that my tube might have not had enough cheel cells because when I tried transfering the cheek cell bead to another tube, it sort of feel apart.So this may have caused me to transfer more of the saline solution rather than my cells.
