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Polymerase Chain Reaction is a useful technique for the detection of genes done through the amplification of specific sites. Alu elements are a polymorphic group of elements found in the primate genome amplified by retroposition. The Alu element help in the polymorphism of the populations. The aim of this study was to use the PCR technique to detect the Alu insertion TPA-25 in cells from student population and its relation with genotype frequency, allelic and ability to establish the Hardy-Weinberg equilibrium. Samples were taken from 81 students. Cells were obtained by mouthwash and Polymerase Chain reaction was done to amplify the TPA-25 fragment. Agarose gel was done for band visualization of 100 bp for absence of the TPA-25 and 400 bp band for the presence of TPA-25. Heterozygous were revealed by the presence of two bands, 100 bp and 400 bp. Genotype information, such as genotype distribution, allelic frequency and Hardy-Weinberg equilibrium was calculated for the population. Heterozygous TPA25 showed the highest value for individual number, genetic frequency percentage, allelic frequency and expected genetic frequency expected. Three different profiles of band were present, 100 bp, 400 bp and 100 bp-400 bp band. Percentage of genetic frequency was between 30.86% and 35.80%, being the expected genetic frequency between 24.01% and 49.98%. Allelic frequencies were 0.52 and 0.49 for presence and absence of TPA-25, respectively. No Hardy-Weinberg was found showing the variability in the population. In conclusion, PCR is a useful technique to study the Alu insertion TPA-25 and its genetic variability in the population.
Keywords: PCR, TPA-25, Alu insertion.
Polymerase Chain Reaction (PCR) is a powerful technique that allow the amplification of the nucleic acid. To detect Alu insertion polymorphism PCR has been used to genotype putative polymorphic loci one by one. PCR technique can be used to determine the presence or absence of the TPA-25 insertion (Hormozdiari, et al, 2011, Schoechetman et al, 1988). Polymorphic Alu insertion is useful for population studies, paternity, gender and ethnicity determinations, and forensic applications (Njoroge et al, 2010).
The Alu family is a group of mobile genetic elements which constitute around 5% of the human chromosome and are present in a high number of copies accumulated in primate during evolution (Roy-Engel et al, 2001). Particularly, TPA-25 is found within an intron of the plasminogen activator gene and it is present in some individuals but not in others. Since it is found in an intron, it is phenotypically neutral and does not affect the expression of the TPA gene. TPA is related with tissue-type plasminogen activator (tPA) and plays and important role in thrombus dissolution. (Ladenvalt et al, 2003).
Using the PCR and the primers for amplification of TPA-25 a genetic profile will be determined, Fragments of 400 bp and 100 bp are produced for homozygous presence of TPA-25 and homozygous absent TPA-25. Upstream primer use is 5’-GTAAGAGTTCCGTAACAGGACAGCT-3’ and the downstream primer is 5’-CCCCACCCTAGGAGAACTTCTCTTT-3’ as oligonucleotide primers flanking the insertion sites.
The aim of this study was to use the PCR technique to detect the Alu insertion TPA-25 in cells from student population and its relation with genotype frequency, allelic and ability to complain with the Hardy-Weinberg equilibrium.
Materials and methods
Cells, used for the DNA template, came from a saline mouthwash using 10 mL of a solution sodium chloride, NaCl (0.9%). The saline mouth-washing procedure is a bloodless and non-invasive. To get a good amount of cell, the mouth should be washed for 30 seconds, while gently chewing the insides of the cheeks. Solution with cells was poured on a falcon tube. One milliliter of the sample was centrifuged, at top speed in a microcentrifuge. After the first centrifugation, another milliliter was centrifuged in the same tube. The supernatant was decanted leaving a 50 µl of the solution used to resuspend it using the vortex. Using a 1.5 mL microcentrifuge tube, a mix of the cells and 200 µl of Chelex (10%) was prepared. Chelex was added so the metals ions were removed to control the PCR inhibition. Mix was incubated at 56 ºC for 10 min, vortexing every 5 min.
PCR Reaction and agarose electrophoresis gel
The PCR reaction was performed using 10 µl of DNA template solution placed on a PCR tube. Fifteen 15 µl of PCR master mix was added.
The reaction master mix for PCR reaction contained, for 20 samples: 262.5 µl GoTaq © G2 Hot Start Green Master Mix (Promega), 26.25 µl upstream primer, and 26.25 µl downstream primer.
PCR conditions were: 94 ºC for two minutes, 30 cycles of 94 ºC for 1 minute, 58 ºC for 2 minutes, 72 ºC for 2 minutes, and 4 ºC hold.
Agarose Gel Electrophoresis
A 2% agarose gel was prepared to visualize the PCR products or fragments. The agarose gel was prepared by dissolving the agarose (2 g) in water, by heating them in a microwave, and cooling to 55 ºC before poured. Five µl of the RedSafe nucleic acid stain were added. Cooled agarose was dispensed on a gel tray with a comb. A ¼ inch deep gel was prepared. Combs and tape were removed and the tray was placed onto the electrophoresis apparatus. A tris/borate/EDTA buffer (10X) was used to cover the gel. Samples, 20 µl, were loaded in each lane. At the same time the 100 bp DNA ladder was loaded to use as molecular weight marker. Electrophoresis was run at 100 volt for 1 h. Gel was visualized using the Gel Imaging System.
Agarose gel analyses
Gels were visualized to detect the PCR bands produced. The electrophoretic pattern is shown on Figure 1. PCR products generated for 8 samples is shown. The 100 bp marker electrophoresis pattern is visualized on lane 1. Two of the eight samples show one band located around 100 bp and one of the sample present two bands around 100 bp and 400 bp. Samples located on lanes 5-8 do not show a defined band.
Genotype Distribution for the samples
The genotype distribution class is shown on table 1. The genotype distribution was determined by counting how many students are homozygous for TPA-25 (+/-), heterozygous (+/-), and homozygous for the absence of TPA-25 (-/-). The values of individual numbers of each class of genotype were between 25 and 29 (Table 1).
Percentages of genotypic frequencies are shown in table 2. All percentages were around 30%, being the highest for the heterozygous the one for TPA-25 (+/-), 35.80%.
Frequency of different alleles.
Using the ratio between the number of copies of each allele and the total number of alleles present, allelic frequencies were calculated. Total number of allele was calculated by counting the homozygous allele state (+/+ and -/-) twice, and heterozygous (+/-) once. Since humans are diploid, the total number of alleles was calculated by multiplying the total number of allele present (81) by two. Allelic frequency was calculated as 2 x homozygotes+heterozygous / Total number of alleles.
For example, when allelic frequency was determined for the presence of TPA insertion (TPA+), the calculation was (2 x 27) + 29/ (81x2) = 83/162 = 0.51. The same calculation was done for the absence of TPA insertion (TPA-), with a result of 0.49.
Expected genomic frequency
Distribution of the genotypes is described by the equation p2+ 2pq +q2 =1. In the Hardy-Weinberg equilibrium, the population is genetically stable, which means that the allelic frequencies remain constant from one generation to another. In the equation, p represents the dominant allelic frequency and q stands for the recessive allelic frequency, being p2 for the dominant homologous phenotype frequency, and q2 for the recessive homologous phenotype frequencies. The heterozygous genotype frequency was represented by 2pq.
Expected genotypic frequencies were calculated for each allelic. Expected genotypic frequency for the presence of TPA (TPA+) calculation was: p2 = (0.51)2 = 0.2601 (27.04 out 100 students, or the 26.01%). For the absence of TPA-25 insertion (TPA-), the expected genotypic frequency was q2, which means (0.49)2 with a result of 0.2401 (23.04 out of 100 students or 23.04%). For the expected frequency genotypic for heterozygous, the calculation was done by 2pq = 0.4998 (49.98 out of 100 or 49.98%).
Observed genotype frequencies vs expected genotype frequencies
Chi square statistical analyses were used to determine the difference between the observed and the expected genotype frequencies. The sum of the (d2/e) factors was used to do the analyses, d representing the deviation of the observed from the expected results and e the expected value. Table 3 presents calculations for chi square analysis. Values of d2/e of 0.037682 for TPA+, 8.806731 for heterozygous, and 0.408205 for TPA- was reported. Chi square value was 9.252618. Table for values of chi-square were used with 1 degree of freedom. This degree is determined by calculating the number of phenotypic class (2 in this study, presence and absence of TPA-25) minus one. P value for the chi square cannot be calculated from the table because it is less than 0.01. This means that 1% of the time, there are deviations.
Polymerase Chain Reaction is a sensitive and sequence specific techniques developed to amplify specific DNA sequences. This technique is used in molecular analysis (Charlieu et al, 1992). Particularly it has been used in the amplification of the Alu sequences including to screen individuals for the presence or absence of the TPA-insertion. (Watkins et al, 2001)
Figure 1 shows the profiles on the agarose gel from the PCR amplification of the Alu insert TPA-25 fragment. The presence or absence of the Alu insertion at the TPA-25 locus on each copy of chromosome 8 determines the size of the amplifications products. Three different profiles can be detected: if the TPA-25 fragment is absent and homozygous, a fragment of 100 bp was present; when there is a homozygous for the presence of TPA-25, a 400 bp fragment will be present; and for heterozygous TPA-25 the two fragments (100 bp and 400 bp) are present. The size of the fragment measured was done using the 100 bp DNA ladder. This marker has 12 bands from 110 to 1517 bp. Results show the presence of one band around 100 bp in two of the eight samples observed in the gel (Fig. 1). One of those profiles also shows a fade band around 400 bp, which can be compatible with another type of genotype; but since is too clear, it was not considered. The presence of this band (100 bp) is related with the absence of the TPA-25. Only one sample shows two bands around 100 bp and 400 bp (lane 4) corresponding to the heterozygous TPA-25. Absence of bands or the presence of bands that are too clear in lanes 5 to 8 can be related with an error during sample preparation, especially the loosing of the cell pellet or the pH of the Chelex solution. Controlling all the process is very important in obtaining accurate results.
Values for genotype distribution show a similar distribution between the 81 students analyzed. All of the genotypes were present, homozygous for TPA-25 (+/-), heterozygous (+/-), and homozygous for the absence of TPA-25 (-/-) in values of 27, 29 and 25 students, respectively. This distribution showed a similarity in genotypes in the population. Twelve of the studied samples did not show any band related with problem in the methodology, as its shown in the electrophoresis profile.
The percentage of genotypic frequencies showed the highest value for the heterozygous TPA-25, being all of them over 30%. Variation of TPA-25 is related to its dimorphic ability having the possibility to be present in some individuals and absent in others.
Allelic frequency was 0.51 and 0.49 for the presence of the TPA-25 insertion and the absence of it, respectively. Allelic frequencies change frequently due to external and internal pressures. Also, between populations there is a frequently allelic changes due some factors as gene flow, and genetic isolation. Alu elements shows a high grade of dimorphism in human population (Batzer et al, 2001).
Expected genotypic frequencies calculated for each allele show variation between the homozygous and heterozygous. Values between 24.01 and 49.98 out of 100 or 24.01% and 49.98% were found being the heterozygous the highest, meaning the prevalent genotype found in the studied population. A comparison between genetic population frequency and expected frequency showed a great variation between them, especially for the heterozygous TPA-25 where values were 35.80% and 49.98% for genetic frequency and expected frequency, respectively. (Table 2). High variation in genetic variation of the population and expected one can be related with errors in procedure that is reflected in 12 individuals showing no band in the electrophoresis gel.
Calculation of the chi square value in the comparison of observed genotype frequencies and expected genotype showed a value of 9.252618. This value was not possible to found in the table used, indicating n values of less than 0.01. These results demonstrate the lack of population of having the Hardy-Weinberg equilibrium at 5% level of significance.
Seven assumptions are considered for the Hardy-Weinberg equilibrium: organisms should be diploid, only sexual reproduction can occur, not overlapping in the population, population size is infinitely large, mating is random, there is no migration and allele frequencies are equal in the sexes. Absence of some of these assumptions mean that it does not comply with the Hardy-Weinberg equilibrium.
In nature, it is difficult for a population to maintain the Hardy-Weinberg equilibrium, since many of the assumptions are hard to fill. Although some assumptions as selection, mutation and small population are not a requirement, the lack of them makes the allele frequencies change over time. So, finding a population having the Hardy-Weinberg equilibrium is very hard in nature. The Hardy-weinberg equilibrium states a constant variation in a population from one generation to another in the absence of disturbing factors. This equilibrium is related with both genotype and allele frequencies which have to remain constant when mating is random and no disruptive circumstances occur, which is difficult in nature populations.
In the case of the TPA-25, some Hardy-Weinberg equilibrium assumptions, such as diploid, sexual reproduction, same allele frequencies for sexes and random mating can be covered, but others cannot.
Concluding this study, PCR technique is a useful technique to detect the Alu insertion PTA-25 using in genotype studies of a population. Also, no Hardy-Weinberg equilibrium was found in the population and it is related with the evolution and variation of populations.
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Lanes 1 100 bp DNA ladder, 2-9 samples.