Alu lab - Alu genetics lab PDF

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Alu genetics lab...

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Human-speific Alu polymorphic insertions

In this study, the Alu allele frequencies of study population were determined through PCR. It was hypothesized that the study population genotype frequencies would be in HardyWeinberg equilibrium and would be similar to the genotype frequencies in the U.S. population data. The study population genotypes were determined through obtaining cheek cells, performing PCR, and then electrophoreses. Chi-squared analysis trends show a P-value less than 0.001 and reject the hypotheses.

INTRODUCTION

Alu elements are referred to as short interspersed elements (SINEs) and are considered retrotransposons also known as “jumping genes” (reviewed in 1). PV92 is a polymorphic human specific element of Alu and is located on chromosome 16. Insertions of Alu have been studied in relation to human population genetics and relationships among these populations (2). In this study, it was hypothesized that the study population genotype frequencies were in HardyWeinberg equilibrium and the observed genotype frequencies will be similar to those collected from U.S. population data. DNA was collected from the cheek cells of a study population. PCR was then carried out using primers specific for PV92. From the results, the genotype frequencies were determined in the study population.

MATERIALS AND METHODS

DNA Preparation. Cheek cells of study subjects were obtained through an oral rinse using saline solution. The oral rinse was then transferred into a micro test tube and centrifuged. Saline supernatant above the cell pellet was discarded. The pellet was then vortexed to resuspend the cells. Resuspended cells were transferred into a screwcap tube containing InstaGene obtained from Bio-Rad (Hercules, CA), and then the contents were vortexed. The tube was placed in a 56°C water bath for ten minutes and vortexed at the halfway point. After the tube was removed from water bath, the matrix solution was mixed. The tube was placed in a 100°C water bath for five minutes then vortexed several times. The matrix was centrifuged for five minutes at 6,000 x g. Polymerase Chain Reactions. PCR tube was obtained and placed in a capless micro test tube. Twenty

μl of supernatant obtained from screwcap tube was transferred into PCR tube. PCR

master mix obtained from Bio-Rad was transferred into PCR tubes containing study subject DNA and control DNA obtained from Bio-Rad. The solutions were mixed by pipetting up and down several times. All PCR tubes were then placed in PTC-200 Thermal Cycler obtained from MJ Research (Waltham, MA). The PCR conditions were pre-denaturation at 94°C for 2 min, followed by 40 cycles of 94°C for 1 min, 60°C for 1 min, and 72°C for 2 min. Final conditions were extra elongation at 72°C for 10 min and then stopdelayed at 4°C indefinitely. Electrophoreses. PV92 XC loading dye was added to each PCR tube. Electrophoresis buffer was poured into electrophorese chamber containing 1% w/v agarose gel in 1X TAE. PCR

samples were loaded into wells and electrophoresed for 45 minutes at 100 volts. Agarose gels were then stained overnight using 1x Fast Blast DNA Stain obtained from Bio-Rad.

RESULTS A representative gel from this study is found in Figure 1. The DNA samples are presented from left to right starting with lane 2 containing the sample DNA size standard, lane 3 containing the control DNA, and the remaining lanes containing study subject DNA. The full set of data obtained from the study group is presented in Table 1. The USA population data obtained from the PV92 PCR Lab manual provided by Bio-Rad was compared to the observed data obtained from the study group. A chi-squared analysis of this data is presented in Table 2. The study population data was tested for Hardy-Weinberg equilibrium. Observed values of p and q were used to determine expected values of p2, 2pq, and q2. A chi-squared analysis of this data is presented in Table 3.

DISCUSSION Through performing PCR and obtaining observed genotype frequencies from the study group, the results show trends that reject the predicted hypotheses. The chi-squared analysis presented in Table 2 demonstrates a chi-squared value of 328.0 and a P-value being > 0.001. This P-value rejects the hypothesis of the U.S. population data being similar to the study population data. The chi-squared analysis presented in Table 3 demonstrates a chi-squared value of 42.02

and a P-value being > 0.001. This P-value rejects the hypothesis of the study population genotype frequencies being in Hardy-Weinberg equilibrium. Possible extensions of this experiment could be to compare U.S. population data sets to data sets from different geographical regions.

ACKNOWLEDGEMENTS

LITERATURE CITED

1. Ray D, Walker J, Hall A, Llewellyn B, Ballantyne J, Christian A, Turteltaub K, Batzer M. 2005. Inference of human geographic origins using Alu insertion polymorphisms. Forensic Science International 153:117-124 2. Batzer M, Stoneking M, Alegrio-Hartman M, Bazan H, Kass D, Shaikh T, Novick G, Ionnou P, Scheer D, Herrera R, Deininger P. 1994. Prov Nattl Acad Sci. 91: 12299-12292.

Figure 1. Representative Agarose gel obtained from this study.a

a

Lane 2: DNA size standard, Lane 3: sample Heterozygote DNA(+/-), Lanes 4-7: study subject DNA (-/-)

Table 1. Full genotypic data set obtained from study group.a

Genotype +/+

Individuals with Genotype 57

+/-

105

-/-

227

a

Observed p value: 0.28, observed q value: 0.72.

Table 2. Chi-Squared analysis of U.S. population data genotype frequencies Genotype Observed Expected Difference (Difference)2/Expected +/+ +/-

57 105

93.4 213.9

-36.4 -108.9

14.2 55.4

-/-

227

81.7

145.3

258.4 328.0a

Total a

With 2 degrees of freedom and a chi-squared value of 328.0, the P-value is > 0.001.

Table 3. Chi-squared analysis of study group data genotype frequencies in Hardy-Weinberg equilibrium. Genotype Observed Expected Difference (Difference)2/Expected +/+ 57 31.12 25.88 21.5 +/-

105

155.6

-50.6

16.5

-/-

227

202.28

24.72

3.02

Total a

41.02a

With 2 degrees of freedom and a chi-squared value of 41.02, the P-value is > 0.001....