Alu Synthesis Lab Report

Determining Genotypic Frequencies for Alu Insertion Polymorphism at the PV92 Locus
Introduction
An Alu element is a short stretch of non-coding DNA found in primates. It gets its name from the single recognition site for the endonuclease Alu I, located near the middle of the Alu element. Alu elements are transposable DNA sequences that copy and insert themselves into new chromosome locations. They are regarded as “selfish DNA” because they do not encode protein and appear to only exist for their own replication. These Alu insertions are significant because each insertion is evidence of a unique transposition event that occurred only once during primate evolution. This means that all primates sharing an Alu allele stem from a common ancestor,
in whom this unique transposition first occurred. Hardy-Weinberg equilibrium states that when certain environmental conditions are met, allele and genotype frequencies in a population will remain constant between generations. Those conditions include the absence of mutation, the absence of migration from other populations, the population is infinite in size, the equal survival and reproduction of all genotypes in the population, and random mating between the individuals of the population. Because natural populations never fully meet all five requirements simultaneously, the Hardy-Weinberg principle serves as a reference point against which the effects of these evolutionary influences can be analyzed. In “Genetic Variation of Recent Alu Insertions in Human Populations,” M.A. Batzer conducted 84 tests for Hardy-Weinberg equilibrium on different human populations, analyzing the Alu alleles for each population. He discovered that there were several Alu variations of African origin, but very few variations of European origin. This indicated that there was a smaller genetic disparity between European populations than that found between other populations groups, like the Africans (Batzer et al., 1996). It was hypothesized that if the Alu genotypes were determined for 28 students in the same biology class, then there would be 9 to 10 students of each genotype. The Alu genotype of the students had no bearing on whether or not they were in the class, so the 28 students were considered to have been randomly selected. Thus, it was predicted that each genotype would be represented equally by the 28 students.
Methods
DNA was obtained from the cheek cells of each student. The students rinsed their mouths with water and then rotated a sterile cotton swab inside their cheek for 45 seconds. The swab was then left to air dry for 15 minutes inside a clean 1.5 mL eppendorf tube. Next, the cotton tip of the swab was removed using a sterilized pair of forceps. The forceps were sterilized by dipping the tip of the forceps into 95% ethanol and then running the tip of the forceps through a bunsen burner flame. The separated swab tip was then placed back into the 1.5 mL eppendorf tube. Using a P200 pipette, 40 µL of 0.2 M NaOH was added to the eppendorf tube. The mixture was heated for 10 minutes at 75 degrees Celsius in a waterbath. Finally, 360 µL of 0.04 M Tris was added, and the contents of the eppendorf tube were mixed by flicking. The swab tip was spun down using a microcentrifuge, and the supernatant (the isolated DNA) was collected. In preparation for the polymerase chain reaction (PCR), 25 µL of collected DNA was first transferred to a 0.2 mL eppendorf tube (the PCR tube). Next, 20 µL of PCR MasterMix was added and mixed with the DNA. This sample was pulsed using the microcentrifuge and then stored on ice. For the actual conduction of PCR, 5 µL of Envision loading dye was first added to the DNA sample. The sample was mixed by flicking and then pulsed using the microcentrifuge. 20 µL of the sample was loaded into the assigned well of a 1% aragose gel. Also loaded onto the first gel were 10 µL of EZ Load Marker, 10 µL of homozygous (+/+) control, 10 µL of homozygous (-/-) control, and 10 µL of heterozygous (+/-) control. The samples were electrophoresed at 100 volts for 2 hours and the results were photographed. Lastly, the bands for each lane of gel were interpreted to determine the students’ genotypic and allelic frequencies for the Alu insertion polymorphism at the PV92 locus. The band patterns for homozygous (+/+), homozygous (-/-), and heterozygous (+/-) were identified, and the bands from the students’ samples were compared to these controls to determine the students’ genotypes. The genotypic frequencies were recorded and the calculations for data analysis were performed.
Results
Of the 28 students, 7 were homozygous (+/+), 9 were heterozygous (+/-), and 12 were homozygous (-/-), as shown in the Genotypic Frequencies of the 28 Student Sample table and PCR Results figures below. Using this experimental data, it was calculated that the frequency of the (+) allele (also known as the p value) was 41.1%. This means that of all the alleles in the sample, 41.1% of them were (+). Each student has two alleles for this gene, so there are 56 alleles total in this sample. 41.1% or 23 of those alleles are (+), meaning that the other 58.9% or 33 alleles are (-). These calculated results are shown in the Allelic Frequencies of the 28 Student Sample table below.

Genotypic Frequencies of the 28 Student Sample
Genotype
Number of Students
Frequency of Genotype
Homozygous (+/+)
7
25.0%
Heterozygous (+/-)
9
32.1%
Homozygous (-/-)
12
42.9%

Allelic Frequencies of the 28 Student Sample
Allele
Frequency
(+)
p = 41.1%
(-)
q = 58.9%

Gel 1 PCR Results
Gel lanes from left to right: marker-(+/+)-(+/-)-(-/-)-sample 1-sample 2

Gel 2 PCR Results
Gel lanes from left to right: marker-sample 3-sample 4-sample 5-sample 6-sample 7
Gel 3 PCR Results
Gel lanes from left to right: marker-sample 8-sample 9-sample 10-sample 11-sample 12

Gel 4 PCR Results
Gel lanes from left to right: marker-sample 13-sample 14-sample 15-sample 16-sample 17
Gel 5 PCR Results
Gel lanes from left to right: marker-sample 18-sample 19-sample 20-sample 21-sample 22
Gel 6 PCR Results
Gel lanes from left to right: marker-sample 23-sample 24-sample 25-sample 26-sample 27
Gel 7 PCR Results
Gel lanes from left to right: marker-sample 28
Discussion
It was hypothesized that if the Alu genotypes were determined for 28 students in the same biology class, then there would be 9 to 10 students of each genotype. Since the 28 student sample was chosen randomly, it was predicted that each genotype (+/+, +/-, and -/-) would be represented equally. However, this was not the case, as 7 students were homozygous (+/+), 9 students were (+/-), and 12 students were homozygous (-/-). Although these were not the hypothesized results, they are logical due to the small sample size. Because the genotypes were recorded for only 28 students, random chance had a large effect on the distribution of genotypes. This is the reason one requirement of Hardy-Weinberg equilibrium is having a population of infinite size. The smaller the sample size, the more of an effect random chance has on the composition (genotypes in this case) of the sample. In M.A. Batzer’s Genetic Variation of Recent Alu Insertions in Human Populations, European-Americans were found to have an Alu frequency of 0.178 (Batzer et al., 1996). The Alu frequency for the 28 person sample (0.411) was 231% that of the European-Americans in Batzer’s study. Firstly, Batzer’s Alu frequency is for European-Americans. The majority of the 28 student sample was Asian-American, a group with a different Alu frequency from that of European-Americans. Secondly, Batzer also used a very small sample size (45 people) in his study. Thus, the Alu frequency of 0.178 is not representative for the actual Alu frequency of European-Americans. It is difficult to compare the Alu frequency of the 28 person sample to published data due to the arbitrary composition of the group. In future research, greater sample sizes will be used to produce more accurate results.
The problem with this experimental setup as well as Batzer’s study was that the sample sizes were too small to provide accurate Alu frequencies. The Alu frequency generated from a sample of 45 people is simply not representative of the entire population of European-Americans. With a larger sample size, the effect of random chance on the results will be greatly reduced. Future experiments will include samples of varying races. Samples based on race would be more meaningful because race the largest impact on Alu frequencies. Furthermore, sampling by race would allow comparison to published data, which also takes samples based on race.

References
Batzer MA, Arcot SS, Phinney JW, Alegria-Hartman M (1996) Genetic Variation of Recent Alu Insertions in Human Populations. J Mol Evol 42:22-29
Batzer MA, Deininger PL (1991) A human-specific subfamily of Alu sequences. Genomics
9:481-487
Batzer MA, Rubin CM, Hellman-Blumberg U, Alegria-Hartman M, Leeflang EP, Stern JD, Bazan
HA, Shaikh TH, Deininger PL, Schmid CW (1995) Dispersion and insertion polymorphism in two small subfamilies of recently amplified human Alu repeats. J Mol Biol 247:418-427
Matera AG, Hellmann U, Hintz MF, Schmid CW (i990b) Recently transposed Alu repeats result from multiple source genes. Nucleic Acids Res 18:6019-6023
Shen M-R, Batzer MA, Deininger PL (1991) Evolution of the master Alu gene(s). J Mol Evol
33:311-320