Amplification of a Non-Coding Region in Chromosome 1, “pMCT118,” and Electrophoresis
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Dr. Kinal
Genetics Lab
Abstract During this experiment, DNA from cheek cells was collected from saline rinses performed by each student in class. The cheek cells were collected by centrifugation which was followed by being resuspended in a Chelex solution. Samples of squamous cells were lysed by being boiled, which resulted in free chromosomal DNA. From here, the samples were introduced to a buffered solution of Taq polymerase, oligonucleotide primers, dNTP and magnesium chloride. The samples were added to a DNA thermal cycler, which went through 30 cycles consisting of 30-second incubation at 94oC, 30-second incubation at 65oC, and 30-second incubation at 72oC. Each sample was pipetted into a well in agarose gel. As an electrical current produced from electrophoresis passed through the agarose gel, the negative DNA samples passed through the gel towards the positive end of the gel. The results from the post-electrophoreisised agarose gel and DNA samples show that 5 individuals within the class were homozygous and 5 individuals were heterozygous at the pMCT118 locus. Two DNA fingerprints were comparatively similar. The others varied to a higher degree.
Introduction
Samples of DNA from each student in genetics lab were collected from saline rinses. Cheek cells washed and collected in the saline rinse were further analyzed. The reason behind this was to amplify a particular non-coding region of chromosome 1. This non-coding region, which is a type of polymorphism known as VNTR, “variable number of tandem repeats,” is pMCT118. This type of VNTR has been described as having a, “…high degree of heterozygosity.” (Lab handout, pg.1) This means that the repeated unit of 16 base pairs is normally different between people, even from individual’s parents. The variation in the number of repeats enables the ability to identify people and establish a DNA finger print. There is a chance that separate individuals may have relatively close DNA fingerprints, though the probability is low. The use of electrophoresis and agarose gel showed the difference in number of non-coding repeats for each student. The distance traveled by alleles, “non-coding repeats in this case,” were observed. Smaller alleles travel farther in the agarose gel. This is because smaller substances travel farther through agarose gel than larger substances. The differentiating number of repeated pMCT118 units between individuals was visible by the bands which traveled across the gel at different distances.
Methods During the experiment, each student in lab poured 10mL of (0.9% NaCl) saline solution in their mouth and rinsed, which helped remove cheek cells. The saline solution was recollected in a paper cup which was used to transfer the rinsed solution into a test tube. The tubes were put in a clinical centrifuge for 10 minutes. This helped collect DNA within the sample at the bottom of the tube. The supernatant from the tube was separated and placed in the paper cup. This was done without moving or disturbing the cell pellet at the bottom of the tube. At this point, two separate methods were
