At the Dolan DNA Lab Center, an experiment was conducted to investigate Alu gene insertion on two alleles at the PV-92 Locus on Chromosome 16. Twenty two students participated in the research in the laboratory. Students washed their mouth with saline solution to extract their DNA sample and gel electrophoresis was used to find their genotype. Six students were reported to have the Alu insertion present on both alleles; ten were reported to have Alu present on one allele and the rest were reported to have no Alu present on any alleles. This experiment was significant because it showed the diversity in the human gene pool. It also showed that majority of the human genome consists of non-coding DNA called "junk genes." It was concluded that Alu insertion is most obvious among Asian population and less in non-Asian. The presence of an Alu on an allele indicates admixture between Asian and non-Asian population.
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Alu genes are believed to be retrosposons that are restricted to the primate branch of the evolutionary tree. Scientists estimate that one million copies of Alu can be found in human, which accounts for 10% of the total genome. Alu are defective Short INterspersed Elements that encode no protein and depend on another intron in the genome called L1 for mobility. L1, which is Long INterspersed Elements, is much longer than the short 300bp nucleotides long Alu. For RNA transcription, Alu relies on the retrovirus, reverse transcriptase enzyme of L1, which has the unique skill of sequencing RNA bases to make complementary strands by cutting and joining the strand at a designated juncture.
During transcription, complex molecules like RNA polymerase III binds to the box on the L-Alu and copies the RNA strand needed as a blue print for other Alu genes. Using reverse transcription, rt copies the information from the RNA template into a DNA strand to make a new Alu element. A DNA polymerase completes the transcription process by forming a new Alu element with repeating sequences on opposite sides of the L-Alu and the R-Alu elements.
The experiment is performed by extracting DNA samples from donors. The DNA sample is placed in a Polymerase Chain Reaction. The PCR amplifies each DNA nucleotide using primers and Tag polymerase as reaction agent. A single DNA strand is denatured to single DNA nucleotides at a temperature close to 100oC. At a low temperature, primers binds to the complementary sequence on each nucleotide to be copied making it easier for the Tag polymerase to locate the targeted spot on the nucleotide. At a slightly higher temperature, Tag polymerase binds to the primers and make a copy of the targeted sequence on the nucleotide.
The PCR cycle is repeated thirty times to have a good amplification of the DNA strands. The DNA sample containing the Alu elements is placed in gel electrophoresis to separate the copied DNA fragments based on their sizes. Although the sample containing the DNA fragments is colorless, scientist adds a blue dye to determine the location of fragments as they move through the gel. In the gel, the DNA strands are separated by length, stained with purple dye, and illuminated with Ultra Violet light. Alu is a Short INterspersed Element of length 300bp. The location of SINEs of length 300bp on maternal or paternal alleles indicates the presence of an Alu gene.
The experiment, if performed on donors' alleles from different regions of the world, can explain diversity within a society, and the history of human migration. Scientists concluded that the frequency of finding Alu on paternal of maternal alleles is higher for an Asian and lower for a non-Asian. The presence of Alu on an allele in a non-Asian society can indicate identity by descent.
The experiment was performed using saline solution, chelex, polypropylene paper cup, micro pipet, micro centrifuge, thermal cycler, and primer. The micro pipet tips were described based on the colors of their ejectors: yellow represent 200 micro liter, light gray represent ultra micro liter, and dark blue represents 1000 micro liter. The students washed their mouth with 10ml saline solution to extract cells from the mouth. The DNA sample were spat into a polypropylene cup. A blue pipette was used to transfer 1ml of sample into 1.5ml plastic tube. The tubes were placed in a centrifuge for the cells to be thoroughly mixed for one minute. After the tubes were removed from the micro centrifuge, the supernatant that accumulated on the surface of the cell pelex were poured into a propylene cup. Then a micro pipette was used to mix the remaining supernatant with the cell pelex by pipetting in and out. The yellow pipette was used to withdraw thirty micro liter of the solution containing the cell pelex suspended in supernatant and put into 100 micro liter of chelex. The solution was shaken before it was placed in a thermal cycler.
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In the thermal cycler, the sample was boiled for ten minutes at 99oC to open the cells, cooled, and then spun for 1min in a centrifuge. The initial amount of 30 micro liter of supernatant was drawn into a 1.5ml plastic tube used for the Polymerase Chain Reaction part. Before the Polymerase Chain Reaction, the yellow pipette was used to draw 22.5 micro liter of PV-92 primer into a Ready-to-Go PCR bead tube. Then the gray pipette was used to draw 2.5 micro liter of DNA sample from the prepared supernatant. The tubes were numbered and the PCR was set for thirty cycles.
The experiment was successful. Twenty two students participated in the experiment and all but one results were shown. A [++, + -, - - ] sign was to indicate the presence or absence of an Alu element on an allele. Out of the twenty two students that participated in the experiment, six were reported to have the [++] sign on the chart, which indicates the presence of Alu elements on the paternal and maternal alleles; ten students were reported to have the [+ -] sign on the chart, which represents the presence of the Alu elements on one of the parental alleles; eleven out of the twelve remaining students were reported to have the [- -] sign on the chart, which indicates the absence of Alu on the parental alleles; one result was not shown. Alu is approximately 300bp long; any element that is more than or less than 300bp is not considered an Alu and indicates the absence of Alu on an allele.
DISCUSSION OF RESULTS: