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This research paper focuses on the distribution of two polymorphic markers: D1S80 and D3S1358. Genomic DNA samples were extracted from buccal cell scrapes of students and analyzed using Polymerase Chain Reaction (PCR) and Polyacrylamide Gel Electrophoresis (PAGE). The study revealed a genotype frequency of 0.0005 for samples 28 and 0.0001 for samples 29. This research suggests the need for further investigation involving DNA samples from populations outside the UK.
Polymerase Chain Reaction (PCR) is a vital technique used for the amplification of specific DNA regions, such as D1S80 and D3S1358. D1S80 is a Variable Number Tandem Repeat (VNTR) locus located on Chromosome 1, characterized by a series of 16 base pair repeats (Huckenbeck et al., 1996).
Each individual inherits two alleles of D1S80, one from each parent. D1S80 is widely utilized in forensic identification due to its informativeness, making it valuable for applications such as paternity testing and population genetics studies (Limborska et al., 2011).
D3S1358, on the other hand, is a Short Tandem Repeat (STR) locus situated on Chromosome 3 at the 1358th locus.
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It comprises two different nucleotide repeats, AGAT and AGAC, with approximately 12 distinct alleles ranging in size from 103 to 147 base pairs.
Polymerase Chain Reaction (PCR) is a fundamental technique in molecular biology with broad applications in gene analysis (Garibyan & Avashia, 2013). PCR finds use in various fields including environmental microbiology, medicine, phylogenetics, forensic science, agriculture, consumer genomics, and genetic research. The PCR process consists of three main stages, as outlined by Garbiyan & Avashia (2013):
This step allows DNA polymerase to copy the single strands.
A sterile swab was rubbed on the inside surface of the cheek. The stick was broken off close to the swab and transferred to a labeled sterile 1.5 ml Eppendorf tube, and the tube lid was closed. 0.3 ml (300 µl) of QuickExtract™ DNA extraction solution was added to the Eppendorf tube containing the swab. It was placed in the Vortex for 15 seconds and then transferred to a 65⁰C heating block and incubated for 10 minutes. Afterward, it was placed in the Vortex for another 15 seconds and transferred to a 98⁰C heating block and incubated for 2 minutes. The resulting samples were stored on ice until used for PCR analysis.
A PCR Eppendorf tube was labeled with the group number, polymorphic marker used, and initials (e.g., 1 D1 NP or 1 D3 NP). The tube contained a master Mix bead, to which the following components were added:
It was essential not to disturb the bead during this process. The solution was pipetted slowly down one of the sides of the tube at an angle. Then, the tubes were immediately placed in the thermal cycler. The cycling parameters were as follows:
| Temperature (⁰C) | Time (seconds) |
|---|---|
| 94 | 240 |
| X 30 cycles: | |
| 94 | |
| 65 | |
| 72 | |
| 60 | |
| X 30 cycles: | |
| 94 | |
| 65 | |
| 72 | |
| 60 | |
| 72 | 600 (final extension) |
| 4 | hold |
| Temperature (⁰C) | Time (seconds) |
|---|---|
| 94 | 240 |
| X 30 cycles: | |
| 94 | |
| 65 | |
| 72 | |
| 60 | |
| 72 | 600 (final extension) |
| 4 | hold |
For the gel pre-run, the polymerized acrylamide gel was removed from packaging, and the plates were rinsed with deionized water to clean them. Tape was placed on the edges to prevent the gel from running off the plate. Any excess polyacrylamide was shaved away from the comb. The position was clearly marked by a permanent marker at the bottom of the wells on the long plate. The comb was removed, and the bottom tape was stripped off. The gel was fixed into the cassette with the longer plate facing out and the well-side on top. The cassette was gently lowered into the buffer tank, ensuring the lid was properly fitted. Approximately 1.5 L of 1X TBE buffer was added to the outer chamber of the electrophoresis apparatus, up to the maximum fill line. The gel was pre-run for 10 minutes at 300V to warm it up, during which time the samples were prepared for loading.
The sample was prepared for electrophoresis by mixing the amplified reaction from the previous practical with 5µl of loading dye. The sample was spun in a micro-centrifuge to bring the contents to the bottom of the tube. A ladder with 50 bp was used. After the pre-run, a pipette was used to flush the well area and load the samples. 10µl of 50 bp ladder was loaded into the middle lane, and then 10µl of each sample was loaded into the respective wells, ensuring no contamination between adjoining wells. The loading process did not take longer than 20 minutes to prevent the gel from cooling. Two samples (D1S80 and D3S1358) were loaded onto the gel per person tested. The lid was replaced on the electrophoresis apparatus, and the leads were connected to the power pack. Sufficient separation was achieved by running the gels at a constant voltage of 300V until the light blue dye front reached the bottom of the gel, approximately 45 – 60 minutes.
After electrophoresis, the buffer chambers were emptied, and the gel clamps were carefully loosened. The gel cassette was removed from the apparatus. The gel was placed on a flat surface, and a steel wedge was used to carefully separate the two plates. The gel was affixed to the longer plastic plate. 100 ml of staining solution was added to a plastic container. The gel was placed onto the gel attached to the plate on top of the solution. The gel was allowed to slide off the plastic plate into the solution using the steel wedge if required. The gel was quite delicate, so caution was exercised. The lid was placed onto the plastic box containing the gel and transferred to the orbital shaker. It was shaken at low speed for approximately 30 minutes. After staining, the gel was carefully removed from the staining solution and placed on the UV trans-illuminator for photography.
| Lanes | Migration Distances (cm) |
|---|---|
| 12 | 3.3 |
| 11 | 2.9 |
| 5 | 3.3 |
| 2.9 | 3.3 |
| 3.5 | 3.5 |
| 3.1 | 3.1 |
| 3.3 | 3.3 |
| 3.2 | 3.2 |
| 3.5 | 3.5 |
| 3.1 | 3.1 |
| 9 | 3.2 |
| 8 | 3.2 |
| 6 | 3.3 |
| 7 | 3.3 |
| 4 | 3.3 |
| Sample | Regression Equation | Base Pair Value | Number of Repeats | Allele Frequency |
|---|---|---|---|---|
| 4 | y = -0.243x + 3.4535 | 501 | 22 | 0.0449 |
| 5 | y = -0.243x + 3.4535 | 401 | 16 | 0.0056 |
| Sample | Base Pairs | D1S80 Genotype | Genotype Frequency |
|---|---|---|---|
| 28 | 501, 401 | 22, 16 | 0.0005 |
| 28 | 501, 401 | 22, 16 | 0.0005 |
| 29 | 562, 447 | 26, 19 | 0.0001 |
| 29 | 562, 447 | 26, 19 | 0.0001 |
The base pairs of each band are used to determine the number of repeats, which is presented as the genotype of the sample in the table. The genotype frequency of each sample is calculated using the Hardy-Weinberg principle. Both samples of 28 have 501 and 401 base pairs, while samples of 29 have 562 and 447 base pairs.
This study investigated the migration patterns of genetic markers D1S80 and D3S180 within a polyacrylamide gel. As outlined in the results section, D1S80 exhibits two bands in each line, whereas D3S180 presents a single band. The presence of two bands on the gel indicates a heterozygous genotype, while a single band signifies a homozygous genotype.
The results have indicated genotype frequencies of 0.0005 and 0.0001. A lower heterozygous genotype frequency suggests that the individual is rarer. Limborska et al. (2011) reported D1S80 alleles with 24 and 18 repeats in non-African samples, which aligns with the findings in this study where D1S80 alleles displayed 22 and 16 repeats, as well as 26 and 19 repeats in different individuals. Similarly, Al Nasser et al. (1996) investigated D1S80 VNTR locus polymorphism in the Kuwaiti population and identified alleles with 18 and 24 repeats. Duncan et al. (1997) noted that the "D1S80 polymorphism is due to variation in the number of repeat units." There are two main types of repeat polymorphisms: VNTR (Variable Number of Tandem Repeats) and STR (Short Tandem Repeats). VNTRs are employed to identify variability in repeat sequences, aiding in genome mapping and genetic fingerprinting. Short Tandem Repeats, or microsatellites, are used to pinpoint specific loci in different DNA samples.
However, there are limitations in this study. All DNA samples were obtained from students residing in the UK. Limborska et al. (2011) conducted similar research with samples from individuals in Europe, Asia, and sub-Saharan Africa, and their findings showed no significant difference in the distribution of D1S80 frequency. Using DNA samples from individuals outside the UK in future studies would enhance global representativeness and improve the research's reliability. Further investigation is required to compare D1S80 genotype frequencies between individuals in the UK and those residing outside the UK.
In conclusion, the study employed D1S80 and D3S1358 alleles to determine genotype frequencies through cheek swab analysis, demonstrating VNTR locus polymorphism in different individuals. This methodology enables successful genetic fingerprinting for human identification and paternity testing.
Genetic Marker Analysis for Human Identification. (2024, Jan 23). Retrieved from https://studymoose.com/document/genetic-marker-analysis-for-human-identification
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