*Corresponding author. email:
Abstract
Alu elements are a group of mobile elements amplified in primate genome through retroposition. One of the Alu elements is the TPA-25. This element presents a high degree of dimorphism within human population, showing different alleles frequencies in different human group. Polymerase Chain Reaction (PCR) technique allow the amplification of part of the nucleic acid, including DNA being detecting by agarose gel. The objective of this study was to compare the genotypes of several individuals by using the Alu insertion detected by the Polymerase Chain Reaction. Cell samples from 82 students were taken. Samples proceed from mouthwash, a non-invasive and bloodless technique. PCR technique was done to detect the production of 100 bp, 400bp, or both bands to determine the homozygous presence, heterozygous presence and absence of PTA-25. Genotype distribution, allelic frequency, and expected genotype were determined and use for the calculation of the Hardy-Weinberg equilibrium. Heterozygous was the highest genotype distribution found and also expected with values around 45% in both cases. Presence of TPA-25 was found in the 29.27% of the population, and absence in the 25,61%. Highest allelic frequency was also found for the heterozygous population. A relation was found between the expected genotype and found genotype. Population did not meet the Hardy-Weinberg equilibrium at 5% level of significance. In conclusion, Polymerase chain reaction is as useful technique to detect genes, particularly for the detection of an Alu insertion polymorphism TPA-25.
Keywords Alu insertion, polymerase chain reaction, polymorphism. TPA-25
Introduction
The Alu family is a group of successful mobile genetic elements, each one having around 300 bp length, and constitutes around 5% of human genome (Stoneking, et al 1997). These elements can be present in a high number of copies reaching a number over a million per genome and they have been accumulating in the genome of human throughout primate evolution (Roy-Engel et al, 2001). Alu elements have amplified in primate genomes through retroposition reaching 500,000 copies per human genome giving an opportunity of unequal homologous recombination (Deininger and Batzer, 1999). One of the Alu elements is the TPA-25 which displayed a high degree of dimorphism within human population, showing different alleles frequencies in different human group (Batzer et al, 1991). TPA-25 is found within an intron of the plasminogen activator gene and it is present in some individuals but not in others. The Alu sequence does not affect the expression of the TPA gene and is phenotypically neutral because is found within an intron.
Polymerase Chain Reaction (PCR) technique can be used to determine the presence or absence of the TPA-25 insertion. PCR is a technique used for the amplification of the DNA or RNA of an organism or gene defect (Schoechetman et al, 1988).
In this experiment, a 400 bp fragment will be amplified, using the PCR technique, when TPA-25 is present and 100 bp fragment when is absent, by using oligonucleotide primers flanking the insertion sites. DNA template is obtained from cell in a saline mouthwash.
The objective of this study was to compare the genotypes of several individuals by using the Alu insertion detected by the Polymerase Chain Reaction.
Materials and methods
Source of DNA template
The cell pellet was obtained by centrifugation of one milliliter of sample at top speed and then repeated with another milliliter. The supernatant was poured off and the cell pellet with about 50 µl was resuspended by vortexing. Cells were transferred to a 1.5 mL microcentrifuge tube with 200 µl of Chelex (10%). Chelex binds metals ions that inhibit the PCR reaction. Contents were mixed and incubated at 56 ºC for 10 min, vortexing every 5 min. The tubes were incubated, after mixing the content, at 95 ºC for 5 min. Tubes were centrifuged for 5 min at top speed and supernatant was transferred to a clean microcentrifuge tube and placed on ice. Supernatant contains the DNA template.
Polymerase Chain Reaction
PCR reaction master mix for 20 samples was composed by:
262.5 µl GoTaq © G2 Hot Start Green Master Mix (Promega)
26.25 µl upstream primer
26.25 µl downstream primer
The upstream primer used was 5’-GTAAGAGTTCCGTAACAGGACAGCT-3’ and the downstream primer was 5’-CCCCACCCTAGGAGAACTTCTCTTT-3’.
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
4 ºC hold
Size of amplification products depends on the presence or absence of the Alu insertion at the TPA-25 locus on each copy of chromosome 8. A unique fragment of 400 bp is present when is homozygous for the presence of TPA-25, a 100 bp fragment for homozygous for the absence of TPA-25 and two fragments (100 bp and 400 bp) for heterozygous.
Agarose Gel Electrophoresis
Results
Presence of bands in the gel
Gels were examined in order to detect the bands produced by the PCR technique. Figure 1 showed the electrophoretic data for the separation PCR products generated for 8 samples. The 100bp marker is also shown. In samples 1 and 5 one band of 400 bp was observed. Four samples (2, 4, 6, and 7) revealed two bands, one at 400 bp and the other one at 100 bp. Samples 3 and 8 revealed one 100 bp band.
Genotype Distribution
Genotype distribution class was determined by counting how many students are homozygous for TPA-25 presence (+/+), homozygous for TPA-25 absence (-/-), and heterozygous for TPA-25 (+/-). Table 1 shows values for genotype distribution. A range between 25.61% and 45.12% of genotype distribution was found, being the heterozygous genotype the highest one.
Allelic frequency.
Allelic frequencies
Allelic frequencies were calculated using the ratio between the numbers of copies of each allele to the total number of allele present. To calculate the total number of allele, homozygous allele state (+/+ and -/-) was counted twice and heterozygous (+/-) was counted once. The total number of allele is calculated by multiplying the total number of alleles present (82) by two, since humans are diploid. Allelic frequency was calculated using the formula
2 x homozygotes+heterozygous
Total number of alleles present
For example, when allelic frequency was determined for the presence of TPA insertion (TPA+) calculation was
(2 x 24) + 37 = 85 = 0.52
(82x2) 164
Allelic frequency for the presence of TPA insertion (TPA+) was 0.52 and for the absence of TPA insertion (TPA -), it was 0.48.
Hardy –Weinberg Equilibrium
When allelic frequencies remain constant from one generation to another then population is genetically stable and it is in Hardy-Weinberg equilibrium.
The distribution of the genotypes is described by the equation
p2+ 2pq +q2 =1,
where p and q represent the allelic frequencies (dominant and recessive, respectively), p2 and q2 the homologous phenotype frequencies (dominant and recessive, respectively) and 2pq the heterozygous genotype frequency.
Calculation of the expected genotypic frequencies was done for each allelic. In the case of the expected genotypic frequency for the presence of TPA (TPA+) calculations was: p2= (0.52)2= 0.2704 (27.04%) (27.04 out 100 students). The expected genotypic frequency for the absence of TPA 25 insertion (TPA-) was q2, that means (0.48)2 with a result of 0.2304 (23.04%) or 23.04 out of 100 students. Finally expected frequency genotypic for heterozygous was 2pq= 0.4992 (49.92%) or 49.92 out of 100.
Differences between observed genotype frequencies and expected genotype frequencies
Using the chi square statistical analyses the difference between the observed and expected genotype frequencies was calculated. This analysis was selected because sample number was small and the analysis take in account that smaller samples deviate more from the expected results than larger samples. Calculations were done as the sum of the (d2/e) factors being d the deviation of the observed from the expected and e the expected value. Calculations for chi square analysis are presented in Table 2. Having a values of d2/e of 0.34175 for TPA= 3.343878 for heterozygous and 0.180625 for TPA- chi square value was 3.866. Using the chi square table with 1 degree of freedom (means number of phenotypic classes (2 in this study, presence or absence of TPA-25 insertion) minus 1, p was between 0.05 and 0.02. This means that 5% of the time deviations.
Discussion
The Alu sequence called TPA-25 is found within and intron of the tissue plasminogen activator gene. This sequence is present in some people but not in others. PCR technique has been used for the amplification of a piece of DNA to screen individuals for the presence or the absence of genes or DNA including the TPA-25 insertion.
Three different profiles were found when PCR technique was done associated with the possible genotypes of the TPA-25 fragment (Figure 1). Bands distance were measured using the 100 bp marker. Migration of the bands is related with the molecular size of the bands, in that way amplification products having more weight migrate slower than the heavy one and stay at the top. Used marker (100 bp DNA ladder), has 12 bands suitable for use as molecular weight standards on agarose gel. Fragments from 110 to 1,517 bp appear when the DNA is digested. The 500 and 1,000 bp have increased intensity to serve as reference point (New England Biolabs)
Phenotypes found was related with homozygous for the presence of TPA-25 (TPA+), having a fragment of 400 bp, homozygous for the absence of TPA-25 (TPA-) with a 100 bp band, and the third one was the heterozygous phenotype having both fragments (100 bp and 400 bp). These results demonstrated the specificity and activity of the primers used for the amplification of the Alu insertion at the TPA-25 locus on each copy of chromosome 8.
Genotype distribution showed the highest value for the heterozygous individuals, 45.12%. Absence of the Alu insertion at the TPA-25 locus was not found in the 25.61% of the studied individuals. The insertion of the TPA-25 is dimorphic that means is present in some individuals and not in others. Due to the presence or the Alu sequence is in an intron, TPA gene expression is not affected and it is phenotypically neutral. This is important when the genotype distribution is studying.
Although the highest allelic frequency (0.52) was found for the presence of the TPA-25 insertion (+) the allelic frequency for the absence was near. In a population, allele frequencies can change over time and they change frequently between populations. Factors, such as genetic isolation, gene flow, mutation, chance, natural and artificial selection are important factors in the variation of the allele frequencies. Batzer et al, 2001 demonstrated that the Alu elements and particularly the TPA-25 displays a high degree of dimorphism within human population, and the frequency of the alleles vary in different human groups.
Expected genotypic frequencies for each allelic showed variation for the presence, absence, and heterozygous, being the heterozygous genotypic frequencies the highest one (49.92%). Expected genotypic frequencies is a definition of one population giving an indication of the most prevalent genotype or least prevalent in the population. Also, it allows the comparison between the obtained results and the expected one in relation with the genotype to determine if the population is in equilibrium. Comparing the expected genotype with the one found in this study values were similar in all genotypes (Table 1). For homozygous values genotype frequency vary from 27.04% to 29.27%, heterozygous 49.92% to 45.12 % and absence of TPA-25 from 23.04% to 25.61% for expected and actual genotype, respectively. This small difference can be related with changes in the experimental process and calculation.
The value for the differences between observed genotype frequencies and expected genotype frequencies, calculated using the chi square statistical analysis, fell in the table between probability value showed values of 1 degree of freedom (means number of phenotypic classes (2 in this study, presence or absence of TPA-25 insertion) minus 1, p was between 0.05 and 0.02. This value showed that the population is not in Hardy-Weinberg equilibrium at 5% level of significance.
Hardy-Weinberg equilibrium is based on seven assumptions as organisms are diploid, only sexual reproduction occurs, generations are not overlapping, mating is random, population size is infinitely large, allele frequencies are equal in the sexes, and there is no migration. When these assumptions or some of them are violated a deviation occurs and expectations are not covered.
Assumptions, such as overlapping of generations, large size of population, no migration generations, are probably not being met by the studied population and maybe cause the non-equilibrium. The random mating is an important assumption. When this assumption is violated the population will not have Hardy-Weinberg proportions.
Other assumptions as selection, mutation and small population size can be violated without losing the Hardy-Weinberg equilibrium, but the allele frequencies will change over time.
In order for a population meet the Hardy-Weinberg population, it is very important to at least fulfill the assumptions, such as that organisms need to be diploid, only sexual reproduction occurs, generation are not overlapping and population mate randomly and do not migrate. So genetic variation need to remain constant from one generation to another and it is very difficult to find a population having any disturb, so the equilibrium can be altered very easy.
Particularly, the TPA-25 polymorphism satisfy some of the Hardy-Weinberg assumptions as diploid, only sexual reproduction occurs, mating is random, and allele frequencies are equal in the sexes.
In conclusion, results demonstrated the ability to use the Alu insertion PTA-25 in determining the genotype characteristics of a population and the ability of using the PCR technique in the detection of genotype distribution, allelic frequencies and the Hardy-Weinberg equilibrium in populations.
References
A Dictionary of Biology. Hardy-Weinberg equilibrium (2004). Encyclopedia.
Batzer MA, Gudi VA, Mena JC, Foltz DW, Herrera RJ, Deininger PL (1991) amplification dynamics of human-specific (HS) Alu family member. Nucleic Acid Res 19: 3619-3623.
Deininger P, Batzer M (1999) Alu repeats and human diseases. Mol Gen Met 67:183-193
New England Biolabs. 100 bp DNA Ladder. http://www.neb.com/products/n3231-100-bp-dna-ladder.
Schoechetman G, Ou C, Jones W (1988) Polymerase Chain Reaction. J infect dis, 158: 1154-1157
Stoneking M, Fontius JJ, Clifford, SL, Soodyall, H, Arcot, SS, Saha N, Jenkins T, Tahir MA, Deininger P, Batzei MA (1997) Alu insertion polymorphism and human evolution:evidence for a larger population size in Africa. Genome Res, 7:1061-1071.
Roy-Engel A, Carroll M, Vogel E, Garber R, Nguyen S, Salem A, Batzer M, Dininger P (2002) Alu insertion polymorphisms for the study of human genomic diversity. Genetics 159:279-290.
Figure legends
d: deviation of the observed from the expected. e: expected value.
