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Genotypes Of The Alu Element

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The Alu insert on the PV92 region is a way to mark both the maternal and paternal history of a population and reflect unique evolutionary events (Nasidze et al., 2001). Although these repeated sequences can help to map out specific characteristics within the human genome, it is still uncertain what their origin and main function is. Alu sequences are located in different places within the human genome; therefore this suggests that there is a significant evolutionary importance. It was from the study of the Alu insertions in the PV92 region that led researchers to track the modern human origin to Africa (Comas et al., 2001).

Alu insertions help to illustrate genetic variability across a population because each Alu insertion that occurs within that population is a unique event in evolution, free of homoplasy (Comas et al., 2001), making the Alu repeats identical by descent from a common ancestor (Batzer, 2002). Alu polymorphisms remain identical when inherited, therefore it is easy to tell where certain insertions came from. Alu elements are markers that are not affected by mutation rates, therefore with mutation and selection ruled out, the analysis of Alu insertions should reflect the progression of relations between gene flow and genetic drift in the past (Garcia-Obreon et al., 2007).

In 2008, in a study conducted by Veerraju, Demarchi, Lakshmi and Venkateswara Rao, it was shown that among five tribal populations studied, there was a high degree of variation among Alu inserts. The significant difference between populations suggests that the Alu insertions can help to map out how related different groups are to each other (Veerraju et al., 2008). Also, in 2007 a study on Iberian Basque populations illustrated a significant difference between populations within the Caucasus, Europe, and North Africa (Garcia-Obregon et al., 2007).

The PV92 region is located on chromosome 16, and the specific Alu insertion we will be studying is dimorphic, as opposed to polymorphic. Alu repeats will usually have more than two phenotypes, however with this Alu sequence within the PV92 region, an individual either has an allele present or they do not have the allele present, and there is no visible phenotype for each genotype (Szczys, 2010). In order to isolate the PV92 target sequence, a polymerase chain reaction (PCR) will be used. PCR is used in vitro, to replicate the specific piece of DNA which is in question. In order for a PCR to take place, a mixture of a DNA template, free nucleotides, magnesium ions, primers, and a buffer is needed (Szczys, 2010). The solution is then run through forty cycles of three different steps including a denaturing step (94ËšC), an annealing step (60ËšC), and an extension step (72ËšC). We will use PCR to amplify our target sequence, so we have more than enough DNA to carry out the experiment.

In this study we are aiming to find if our population is Hardy-Weinberg Equilibrium (HWE). Traditionally there are five assumptions a population must meet in order to be considered for HWE. These five assumptions are: no natural selection, large population, no gene flow, no mutation, and random mating (Freeman, 2008). If a population has all five of these characteristics, it is assumed that no evolution is taking place. The purpose of this particular experiment is to use the Hardy-Weinberg Equilibrium approach to determine if our data samples taken within the lab sections were experiencing some form of change, or if we can accept our null hypothesis that the population will be in HWE. We can assume that our data will show signs of evolution because our sample size is very small and can reflect significant deviation. The three genotypes being investigated are homozygous positive, (++) which has the Alu insert present on both alleles, heterozygous (+-) which has the Alu insert present on one allele and not the other, and finally homozygous negative (- -) which is lacking the Alu insert on both alleles.

Materials and Methods

Two hairs were collected, trimmed to 2-3cm containing a coat of epithelial cells around their base and placed in a screw cap tube containing 200µl of InstaGene matrix plus protease. The tubes were incubated for five minutes at 56°C, vortexed, and then incubated again for another five minutes. The tubes were then placed in the boiling water bath (100°C) for five minutes. The tubes were vortexed and then centrifuged at 6,000 x g for five minutes. Then 20µl of the supernatant was transferred into the bottom of a PCR tube and 20µl of the master mix was added. The solution was mixed with a pipette and tubes were then placed in the MyCycler thermal cycler for 40 cycles of amplification in approximately 3 hours.

The PCR samples were removed from the thermal cycler and 10µl of PV92 XC loading dye and 10µl of SYBR gold dye was added to each of the PCR tubes and mixed gently. Then 20µl were taken from each PCR tube and loaded into individual wells within the previously prepared agarose gel. Molecular weight standards and DNA controls (+,+), (+,-), (-,-) were loaded next to the samples for comparison purposes. The gel was then run at 100 volts for thirty five minutes and then 200 volts for an additional forty five minutes. The distance the samples traveled was measured by a photograph taken with UV light.

To see if the population was in Hardy-Weinberg Equilibrium we used the equation p2 + 2pq + q2 = 1.0 to determine phenotype frequencies. To determine expected numbers of students as well as the allele frequency of p we used and to determine the allele frequency of q the equation was used. The allele frequencies produced from these equations can be tested by p + q = 1.0. Using the Chi-Square equation, we compared the Chi-Square statistic to a critical value. If the P value is greater than 0.05, the hypothesis that the population is in Hardy-Weinberg Equilibrium can be accepted.


The data was obtained by counting the number of students showing each of the three possible genotypes. The DNA standard used to reflect upon the DNA samples consisted of two homozygous (++) (- -) and a heterozygous (+-) sample. The bands of students possessing two chromosomes with the insert were 941 base pairs long, and those students lacking the insert on both chromosomes had bands at 641 base pairs long. Students who showed a heterozygous genotype had both a band at 941 base pairs and a band at 641 base pairs (Figures 1 and 2). Of the 18 students within the population, one was homozygous positive, eight were heterozygous, and nine were homozygous negative (Table 1).

The allele frequencies for p and q were 0.28 and 0.72 respectively and the phenotype frequencies were 0.08 for homozygous positive, 0.40 for heterozygous, and 0.52 for homozygous negative. The Chi-Square statistic was calculated to be 0.142. The Chi-Square statistic was found to have a P value between 0.95 and 0.90 at two degrees of freedom.

Figure 1. The gel for the PV92 insert for the AM lab section represents homozygous negative (wells 2, 8, 15) and heterozygous (wells1, 5, 7, 9, 11, 12, 14) traits for the Alu. 10 student samples (5 failed) are compared to the three DNA standards run at the right side of the Gel. One band at 641 base pairs represents homozygous negative, at 941 base pairs homozygous positive and two bands indicate a heterozygous trait.

Figure 2. The gel for the PV92 insert for the AM lab section represents homozygous positive (well 8), homozygous negative (wells 3, 4, 5, 6, 7, 11) and heterozygous (well 2) traits for the Alu. 7 student samples (4 failed) are compared to the three DNA standards run at the right side of the Gel. One band at 641 base pairs represents homozygous negative, at 941 base pairs homozygous positive and two bands indicate a heterozygous trait.


Based on the data collected, it is evident that we can accept our null hypothesis and assume that our population is in fact in Hardy-Weinberg Equilibrium. This was our null hypothesis because our population did not meet all five assumptions of HWE and we expected some form of significant deviation to be present. Our sample was very small, in that only 18 students were involved in the genotyping whereas a large sample generally consists of 1,000 or more individuals. So although our particular study was proven to be within Hardy-Weinberg Equilibrium, in larger populations across the world, the statistics surrounding Alu insertions should all show signs of evolution and genetic drift in order to map out relatedness among populations.

Although Alu inserts can be used to determine certain aspects of evolution, the origin and function of the inserts is uncertain (Comas et al., 2001). Since they can tell us so much about the evolutionary progress of populations, once the function of Alu inserts is figured out they may be able to aid in research on certain diseases which they have been previously linked (Szczys, 2010). It could be very beneficial to understand why some individuals posses the inserts and some individuals do not. Are there health advantages to having the insert or lacking the insert? And if so, are the evolutionary trends we see based on these advantages and factors for selection? It is obvious that Alu inserts give a lot of information concerning patterns within the human genome, but there is much more left to discover about them.

Literature Cited

Batzer, M., Deininger, P. (May 2002) Review of Alu repeats and human genomic diversity. Nature Reviews

Genetics. 3: 70-79

Batzer, Mark A., Arcot, Santosh S., Phinney, Joshua W., E. et al. 1996. Genetic Variation of Recent Alu Insertions in Human Populations. Journal of Molecular Evolution, 42:22-29.

Comas, D., Plaza, S., Calafell, F., Sajantila, A., and Bertranpetit, J. 2001. Recent insertion of an Alu element within a polymorphic human-specific Alu insertion. Molecular Biology Evolution, 18.1: 85-88.

Freeman, Scott. Biological Science. 3rd ed. San Francisco: Pearson, 2008. Print.

Garcia-Obregon, S., Alfonso-Sanchez, M.A., Perez-Miranda, A.M., de Pancorbo, M.M., and Pena, J.A. 2007. Polymorphic Alu insertions and the genetic structure of Iberian Basques. Hum Genet. 52:317-327

Nasidze, I., Risch, G., Robichaux, M., et al. 2001. Alu insertion polymorphisms and the genetic structure of human populations from the Caucasus. European Journal of Human Genetics, 9: 267-272.

Szczys, P. 2010. Molecular Genotyping for PV92. BIO 230 Laboratory Manual. Biology Department, Eastern Connecticut State University, Willimantic, CT.

Veerraju, P., Demarchi, D.A., Lakshmi, N., and Venkateswara Rao, T. 2008. Insertion/ Deletion Polymorphisms in Indian Tribal Populations. Int J Hum Genet, 8(1-2): 75-83

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