Alu Insertions

Discussion: Coded by a polymorphic gene, Alu insertion is found within an intron of the tissue plasminogen activator (tPA), which is lacking in ancient DNA sequences (Batzer et al, 1994). In this experiment, the lining of cheek cells of 105 individuals were tested for the presence/absence of Alu insertion titled tPA-25 by using PCR amplification. Using oligonucleotide primers to border around a tPA-25 insertion site, PCR products were amplified for 400bp fragments which were positive for Alu and 100bp fragments which were absent. The observed genotypes expressed in Table 1 (Appendix A) contain 17 individuals of 400bp fragment, 27 individuals of 100bp fragment, and 61 individuals of both from a party of 105. These observed values were detected using an Orange G dye running through an agarose gel. With these results, the allele frequency was determined at 0.4524 for tPA-25 present (+) and 0.5476 for tPA-25 absent (-) (Allele frequency calculation, Appendix B). Furthermore, the computed value for the chi-squared test revealed that the alleles coding for tPA(+) and tPA(-) insertion are in Hardy-Weinberg equilibrium as the null hypothesis could not be rejected (Chi-squared test, Appendix A).

For something to be in Hardy-Weinberg equilibrium five conditions must be met: random mating, no mutations, no migration, infinite size/population and no sexual/natural selection. Since the equilibrium is met in this experiment, it suggests all 5 conditions apply to the population. Mating is random and no natural selection occurs as the Alu insertion genotypes have no phenotypes nor give any particular advantage to the individual (Lewis, 2007). The population is considered infinite because there are enough individuals once the class data is combined. Mutations occur randomly, but the fact that it is in Hardy-Weinberg equilibrium means that if mutations do occur the rate at which it does is so minimal that it has little to no affect. Migration most likely occurred among the parental generation, but because the equilibrium was kept, it suggests that no one allele were disproportionally added or removed. Thus, with the five conditions met, Hardy-Weinberg equilibrium is sustained on all alleles.

Some individuals’ PCR product was undetectable when examined under a transilluminator. Multiple factors can attribute to this. One possibility is that an adequate amount of centrifugation and boiling did not occur to lyse the nuclear membrane, stopping the primer from attaching to the DNA, resulting in no PCR product. Another factor is that mutations or contaminations could have prevented the primer from attaching, meaning no amplification to detect the product. Lastly, deficient amounts of Chelex 100 and/or master mix could have been used which are used to stop PCR inhibitors and provide reagents necessary for amplification, which would lead to no band (Scortichini et al, 1998). Appendix A

Table 1: Observed frequencies of three genotypes of tPA-25 insertion. Observed Genotype100bp/100bp
(tPA-25 absent/tPA-25 absent)400bp/100bp
(tPA-25 present/tPA-25 absent)400bp/400bp
(tPA-25 present/tPA-25 present)

Frequency
276117

Chi-Squared Test
Null Hypothesis: The alleles that encode for tPA-25 insertion are in Hardy-Weinberg equilibrium, therefore there is no significant difference between observed and expected values. Alternate Hypothesis: The alleles that encode for tPA-25 insertion are not in Hardy-Weinberg equilibrium, therefore there is a significant difference between observed and expected values. Table 2: Chi-squared test of observed and expected values for the three possible TPA-25 genotypes. GenotypeObserved FrequencyExpected Frequency(O-E)2/E

100bp/100bp
(tPA-25 absent/tPA-25 absent)27320.7813
400bp/100bp
(tPA-25 present/tPA-25 absent)61521.5577
400bp/400bp
(tPA-25 present/tPA-25 present)17210.7619

Chi square value = 3.1009
p = 0.0783
df = 1
At a critical value of 3.84 and significance level of 0.05, the...