Allele Synthesis Lab Report

Determining Allele Frequencies of the PV92 Alu Element using DNA Isolated from Human Cheek Cells and PCR Amplification

Background
Alu elements are the most abundant repetitive elements in the human genome that have mobilized throughout primate genomes by retrotransposition over the past 65 million years ago from a 5’ to 3’ fusion of the 7SL RNA gene, to reach the present number of more than one million copies. Over the last few years, several lines of evidence demonstrated that these elements modulate gene expression at the post-transcriptional level in at least independent manners. They have been shown to be involved in alternating splicing, RNA editing and translation regulation (1). These elements have also been proposed to have
a number of functions in the human genome and continue to amplify at a rate of about one insertion every 200 new births (2). The goal of this experiment was to determine the allele frequencies and genotypes using DNA isolated from human cheek cells in a group of 30 individuals in Vermont.
Methods
Isolation of DNA from Cheek Cells
Ten millilitres of 0.9% saline solution was put in my mouth for 30 seconds to rinse cheek pockets to isolate as much DNA from my cheek cells. After spitting the saline solution into a paper cup, the saline solution was swirled gently to mix cells that may have settled to the bottom of the cup. A micropipette was used to transfer 1500 µL of the solution into a 1.5-mL microcentrifuge tube. The sample tube was then placed in a balanced configuration in a microcentrifuge and was spun for 90 seconds at full speed. The supernatant was then removed from the microcentrifuge tube back into the paper cup, while not disturbing the cell pellet at the bottom on the tube. The yellow micropipette was then used to resuspend 30 µL in the remaining saline by pipetting in and out. Thirty µL were then withdrawn and added to a new 1.5 mL tube containing 100 µL of Chelex. The 1.5-µL tube was then placed into boiling water for 10 minutes. After 10 minutes of boiling, the 1.5 µL tube was shaken for 5 seconds using a vortexer on the touch setting. The tube was placed back into a microcentrifuge and spun for another 90 seconds. The sample was then stored on ice until it was needed for amplification.
Amplification of PV92 Locus by PCR
A PCR bead in a 0.2 mL tube was combined with 22.5 µL of PV92B primer/loading dye mix and gently mixed. After about a minute, the bead dissolved and 2.5 µL of my DNA was added directly to the primer/loading dye mix. The sample was then stored on ice to prepare for thermal cycling. The PCR tube was placed with other samples in an ABI Veriti thermal cycle of 30s at 94 °C, 30s at 68 °C and 30s at 72 °C to run for 30 cycles. After cycling was complete, the PCR tube was stored at -20 °C until gel electrophoresis.

Analysing PCR Products by Gel Electrophoresis
The ends of the gel-casting tray were sealed with rubber dams and a well-forming comb was inserted.
Fifty milliliters of 1.5% agarose solution was swirled and inserted into the microwave for 1 to 2 minutes until the agarose solution was clear. The solution was put into a cooling bath of 60 °C for 15 minutes. After cooled, the agarose was poured into the prepared gel-casting tray for 30 minutes until it became solid. The well-forming comb was then removed and 1X TBE buffer was added to fill in wells, to create a smooth buffer surface. Using a micropipette 25 µL of my dye sample mix was added to the well. The gel was run at 130 V for 30 minutes. The gel was stained for 10 to 15 minutes using ethidium bromide. The gel was viewed using UV transillumination and a photograph was taken of the gel.
Analysis of Genotype and Allele Frequency In Whole-Class Data
Each student’s genotype of +/+, +/-, and -/- were counted and put into a genotype frequency equation of to get a percentage. The Hardy-Weinberg equation was then used to determine the genotypes frequencies that were expected in a population at equilibrium. The class data was entered into the Allele Server database (http://www.bioservers.org) where a chi-square test was used to compare observed genotype frequencies with those predicted by the Hardy-Weinberg equation. The data was then compared to genotype frequencies in world populations, where the data were compared in pie charts and several comparisons were
done.

Results
Individual DNA Isolation and PCR of PV92 Locus
Amplification of DNA by PCR and gel electrophoresis by PCR amplification was successful. The bands were shown very clearly for each sample. The sample showed that both bands were homozygous negative (Figure 1).
Figure 1. Electrophoretic gel showing bands from Mariah Stearns in lane 2 and bands from Brandi Gervais is lane 3.

Genotype and Allele Frequency for Whole-Class Data
The whole-class data showed that out of the twenty-six participants no one had the genotype of homozygous +/+, 31% was heterozygous, and 69% were homozygous -/- (Table 1). The Chi square value was 86%, which was close to expected considering the p-value of 0.3526 (Table 1). The class data was then compared to two different world populations. The first was German, showing the two populations have a total of 1 individual with the +/+ genotype, 19 with the +/- genotype and 71 with the -/- genotype (Table 2). The second comparison was to Chinese, which showed the two populations had a total of 37 individuals with the +/+ genotype, 20 with the +/- genotype and 19 with the -/- genotype (Table 3).
Table 1. Chi-square test whole-class Data
Chi-square: 0.86
P-value: 0.3526 Observed
Expected

Total samples: 26
+ Alleles: 8
- Alleles: 44
Total samples: 26
+ Alleles: 8
- Alleles: 44
+/+
0.00;
0.02
+/-
0.31;
0.26
-/-
0.69;
0.72

Table 2. Chi-square test whole-class data vs. German

Chi-square: 2.47
P-value: 0.2914 German
Alongi Cell-Molecular 2013 Total samples: 65
+ Alleles: 13
- Alleles: 117
Total samples: 26
+ Alleles: 8
- Alleles: 44
+/+
0.02;
0.00
+/-
0.17;
0.31
-/-
0.82;
0.69

Table 3.
Chi-square test whole-class data vs. Chinese

Chi-square: 50.46
P-value: 0.0000 Chinese
Alongi Cell-Molecular 2013 Total samples: 50
+ Alleles: 86
- Alleles: 14
Total samples: 26
+ Alleles: 8
- Alleles: 44
+/+
0.74;
0.00
+/-
0.24;
0.31
-/-
0.02;
0.69

Conclusions
Amplification of DNA by PCR and gel electrophoresis of PCR amplification products was successful for the individual data collected. The null hypothesis matched the observed data that was received using the allele sever. The class data showed that for 26 participants no one had the genotype of homozygous +/+, 31% was heterozygous, and 69% were homozygous -/-. The Chi square value was 0.86, which was close to expected considering the p-value of 0.3526. The whole-class data compared to the German was similar in genotypes, but almost opposite genotypes to the Chinese.
References
1. Hasler J, Strub K: Alu Elements as Regulators of Gene Expression. Nucleic Acid Res. 2006, 19:5491-5497
2. Deininger P, Batzer M: Alu Repeats and Human Disease. Molecular Genetics and Metabolism. 1999,
67:183-193.