Exploring VNTRs and PV92: DNA Fingerprinting & Hardy-Weinberg Deviation

Abstract

In this study, we collected DNA samples from oral cavity epithelial cells of a student population. We amplified variable number tandem repeats (VNTRs) D1S80, D17S30, and apoB, as well as the Alu element PV92 using polymerase chain reaction (PCR) and visualized the results through agarose gel electrophoresis. Our objectives were to explore the potential applications of VNTRs in DNA fingerprinting and to investigate whether PV92 adheres to the Hardy-Weinberg principle.

Our findings indicate that PV92 does not conform to Hardy-Weinberg equilibrium (p = 0.

010) in our tested population of 87 individuals. We attribute this deviation to the relatively small population size and limited ethnic diversity within the sample. While VNTRs show promise as tools for creating unique DNA fingerprints, our study suggests that further elaboration is necessary, particularly due to the small sample size (n = 9). We recommend expanding the sample sizes in future experiments to enhance the robustness of our conclusions.

Introduction

This protocol aims to demonstrate how variable number tandem repeats (VNTRs) can be employed to generate unique DNA fingerprints for individuals and to assess whether the Alu element PV92 adheres to the principles of Hardy-Weinberg inheritance.

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Our methodology involves the collection of oral cavity epithelial cells, DNA extraction, amplification of specific sequences, and subsequent analysis through gel electrophoresis.

The first step in our procedure involves the preparation of DNA through thermal lysis, where heating disrupts cell membranes, denatures membrane-stabilizing proteins, and releases DNA (Shehadul Islam, Aryasomayajula et al. 2017). Subsequently, DNA is amplified using PCR, with forward and reverse primers flanking the target region and a thermostable DNA polymerase synthesizing new DNA strands using provided deoxynucleotides.

This process involves cycles of DNA denaturation, primer annealing, and DNA polymerization, occurring at temperatures exceeding 94°C, between 50-70°C, and 70-80°C, respectively (Lorenz 2012).

The separation of DNA is achieved through agarose gel electrophoresis, wherein DNA is placed in a porous agarose gel with a buffer solution. Due to its negative charge, DNA migrates towards the positive electrode upon the application of voltage. The distance traveled is inversely proportional to the logarithm of the DNA's weight, causing larger fragments to migrate less than smaller ones (Lee, Costumbrado et al. 2012).

Variable number tandem repeats (VNTRs) are short DNA sequences ranging from 16 to 64 base pairs, characterized by variable copy numbers. Enzymatic restriction cutting of VNTRs leads to varying fragment sizes among individuals (Blanco and Blanco 2017). These sequences are often located in intergenic regions (Pitt and Barer 2012) and are non-coding, exempting them from the effects of natural selection. Combining multiple VNTRs can create a unique DNA fingerprint for an individual (Blanco and Blanco 2017). The VNTRs examined in this study include D1S80, D17S30, and apoB.

Alu elements are short interspersed DNA repeats that have integrated into the human genome through retro-transposition. Some of these elements are neutral in terms of natural selection, and their positions in the human genome exhibit polymorphism. The Alu element PV92 is both selection-neutral and polymorphic (Comas, Plaza et al. 2001); hence, it is expected to adhere to the Hardy-Weinberg equilibrium.

The Hardy-Weinberg principle posits that, in the absence of evolution, gene shift, or mutation under Mendelian genetics, allele frequencies remain constant between generations. Genotype frequency distribution follows a multinomial expansion for alleles, summing up to one. For two alleles, denoted as p and q, this implies that allele frequencies are equal to p and q, and genotype frequencies follow the equation (Stern 1943):

p² + 2pq + q² = 1

Materials and Methods

The experiment was conducted following the guidelines outlined in the BI2015 2019 lab booklet, with specific modifications. Notably, alterations were made to the durations of the PCR cycles, and these adjustments are detailed in Table 1 below. Additionally, the loading dye used for gel electrophoresis was diluted to a 5X concentration instead of the standard 10X, leading to changes in the volumes loaded. Specifically, 16 µL of the PV92 sample was loaded, along with 4 µL of dye. For the three VNTR samples (ApoB, D1S80, and D17S30), 5.33 µL of each was combined and loaded, along with 4 µL of dye.

Results

In the electropherogram displaying VNTRs D1S80, D17S30, and apoB the alleles of PV92 were enumerated (refer to Table 2), and their respective fractions were employed as a basis for calculating the expected genotype fractions in accordance with the Hardy-Weinberg principle (see Table 3). For complete gel images of PV92, please refer to Supplement 2.

Genotypes were also tallied (see Table 3) and subsequently compared to the expected genotypes derived from allele frequency data (Table 2) using the χ² test, yielding a p-value of 0.010.

Discussion

Results clearly illustrate that all the VNTR bands in the gel are distinct from one another, and in cases where two individuals share a band, there is another unique band to differentiate them. Since we examined three unique VNTRs, the possibility existed for each lane to exhibit between 1 and 6 bands in total. In the extreme scenario, all the VNTRs could overlap entirely, while in the opposite extreme, an individual could be heterozygous for all three VNTRs, resulting in 2 bands per VNTR. It is important to note that the limited presence of bands in the gel could be indicative of PCR failures. This could be attributed to the InstaGene matrix, which was utilized to protect DNA during purification but may have inhibited the PCR polymerase cofactors. Despite the potential failures, the technique shows promise for creating genomic fingerprints due to the observed individual variations. Notably, the closest band to mine (lane 14, 296 bp) was in lane 10, measuring 270 bp (for formula and standard curve details, refer to Supplement 1). While this may not be statistically significant due to the small "successful" population size of 9, it remains a promising finding. Expanding the sample size, possibly to the scale of an entire city, could provide more conclusive results.

Comparing the observed PV92 genotypes (Table 3) with the expected genotypes calculated from allele frequency data (Table 2) reveals significant discrepancies (p = 0.010). This contradicts the findings of Comas, Plaza et al. (2001). The primary contributing factor to these disparities may be the relatively small sample size (n = 87) employed in our study. Furthermore, the population under investigation is not ethnically homogenous. Comas, Plaza et al. (2001) suggests that the PV92 insertion is more prevalent among East Asians and Amerindians. Given that our tested population consists mainly of Northern Europeans, with some East Asians, the genotype frequencies were skewed. Achieving homogeneity or complete heterogeneity within the population could potentially yield different results. Therefore, we recommend increasing the sample size, possibly to encompass an entire city, to mitigate these effects and obtain more reliable data.

Conclusion

The extraction and visualization of VNTRs D1S80, D17S30, and apoB from a student population (n = 9) have demonstrated the potential of VNTRs as excellent candidates for DNA fingerprinting. These VNTRs produced distinct and unique patterns for each individual within the tested population. However, it is important to note that this conclusion may evolve with the utilization of a larger and more diverse population than the one examined in this study.

Analyzing the genotype frequencies of the Alu element PV92 in a student population (n = 87) has revealed that it deviates from the Hardy-Weinberg equilibrium (p = 0.010). This deviation is attributed to the small size of the population and its lack of ethnic homogeneity. Expanding the sample population size is recommended to mitigate the effects of this skewing.

References

• Blanco, A., & Blanco, G. (2017). Chapter 21 - The Genetic Information (I). Medical Biochemistry. A. Blanco and G. Blanco, Academic Press: 465-492.
• Comas, D., Plaza, S., Calafell, F., Sajantila, A., & Bertranpetit, J. (2001). Recent Insertion of an Alu Element Within a Polymorphic Human-Specific Alu Insertion. Molecular Biology and Evolution, 18(1), 85-88.
• Lee, P. Y., Costumbrado, J., Hsu, C.-Y., & Kim, Y. H. (2012). Agarose gel electrophoresis for the separation of DNA fragments. Journal of Visualized Experiments: JoVE(62), 3923.
• Lorenz, T. C. (2012). Polymerase chain reaction: basic protocol plus troubleshooting and optimization strategies. Journal of Visualized Experiments: JoVE(63), e3998-e3998.
• Pitt, T. L., & Barer, M. R. (2012). Chapter 3 - Classification, Identification, and Typing of Micro-organisms. Medical Microbiology (Eighteenth Edition). D. Greenwood, M. Barer, R. Slack, and W. Irving. Edinburgh, Churchill Livingstone: 24-38.
• Shehadul Islam, M., Aryasomayajula, A., & Selvaganapathy, P. R. (2017). A Review on Macroscale and Microscale Cell Lysis Methods. Micromachines, 8(3), 83.
• Stern, C. (1943). THE HARDY-WEINBERG LAW. Science, 97(2510), 137-138.

Supplement 1: Calculation of VNTR Fragment Sizes

To determine the sizes of VNTR fragments, we utilized image analysis software, ImageJ, and reference to the DNA ladder present in the gel. The creation of a standard curve for DNA migration was pivotal to this process. Raw data detailing travel lengths and their corresponding fragment sizes are provided in Table 4.

Supplement 2: PV92 Electropherograms

This supplement comprises the electrophoresis gel images that were analyzed during our study. Heavier fragments in these images indicate the presence of the PV92 allele. The legend for the images is as follows: (-/-) denotes homozygous non-PV92, (+/-) represents heterozygous individuals, and (+/+) signifies homozygous PV92 individuals. Additionally, "L" denotes ladder or control samples, while non-labelled lanes were not taken into consideration during the analysis.