Variable number of tandem repeats (VNTRs) are short repeat sequences that are positioned as a string along a chromosome. The number of repeat sequences commonly varies among individuals hence VNTRs are also described as polymorphic. VNTRs are a type of minisatellite repeats, composed of 14 to 100 nucleotides that are repeated from 4 to 40 times at one time. In this laboratory exercise, total genomic DNA was extracted from cheek squamous cells in order to generate a significant number of copies of a specific non-coding region from chromosome 1 which harbored the VNTR pMCT116.
The number of copies of the VNTR pMCT116 was then separated on an agarose gel in order to determine the number of copies in each chromosome. The laboratory exercise has shown that each individual’s sample that was used in the experiment carried a different number of repeats in the VNTR locus pCMT116. Of the 4 samples analyzed, 2 samples indicated similar banding patterns. RESULTS. ISOLATION OF CHEEK SQUAMOUS CELL TOTAL GENOMIC DNA Squamous cells from the cheek lining were collected through oral gargling of 0.
9% isotonic saline (NaCl) solution for 10 minutes. Total genomic DNA was extracted from the pelleted cells through the use of 10% Chelex solution, supplemented with resin beads. The cell suspension was then incubated in 100oC for 10 minutes after which the suspension was exposed to rapid freezing. The suspension was spun separate any cellular debris from the organic layer of the solution. AMPLIFICATION OF VNTR pMCT116 AND SEPARATION OF PCR PRODUCTS
The pMCT116 VNTR was amplified using the following cycle conditions for each step of the polymerase chain reaction: denaturation step at 94oC for 30 seconds, annealing step at 65oC for 30 seconds and extension step at 72oC for 30 seconds. Amplification was performed for 30 cycles. The 30th cycle was kept for an extra 10 minutes in order to accumulate as much PCR products from the reactions. Approximately 25 l of the PCR product mixed with loading dye was loaded onto each well of a 1. 5% agarose gel. As molecular weight marker, pBR322 digested by BstNI was loaded into another lane of the gel.
Agarose gel electrophoresis was performed at 130 volts for 20 to 30 minutes, or until the cresol red dye had migrated approximately 50 mm from the wells. The gel was viewed under ultraviolet transillumination optics and photodocumented as shown below: Figure 1. 1. 5% agarose electrophoresis of cheek squamous cell-derived VNTR pMCT116 (lanes 1 to 4). Analysis of the banding patterns generated after amplification of the VNTR pMCT116 showed that there are approximately 3 types of polymorphisms in that particular chromosome 1 locus.
As shown in lane 1, there is only one band existing, while in lanes 2, 3 and 4, two bands were observed in each lane. The bands located in lanes 3 and 4 were identical, suggesting that the same kind of polymorphism existed in these two samples. As for lane 2, a different polymorphism existed that also generated two bands. The two bands present in lanes 2, 3 and 4 showed that there are two alleles existing in VNTR pMCT116. The single bright band in lane 1 indicates that there is only one allele existing for VNTR pMCT116 in the sample.
The brightness of that single band indicates that there are two copies of the same molecular weight in that same area. The approximate sizes of the PCR products were 1058 bp and 929 bp. DISCUSSION. The employment of the polymerase chain reaction has allowed the amplification of the VNTR pMCT116 that facilitated the analysis of the number of repeats that were present in the chromosome 1 locus. The laboratory exercise has shown that each individual’s sample that was used in the experiment carried a different number of repeats in the VNTR locus pCMT116.
Of the 4 samples analyzed, 2 samples indicated similar banding patterns. Variable number of tandem repeats (VNTRs) are minisatellites that are distributed across the entire human genome (Lindstedt et al. , 2003). The variation in the number of repeat units in each VNTR provided a reliable and powerful basis in identifying every single individual from a huge population. VNTRs are generally employed in DNA fingerprinting or DNA profiling analysis, which is a modern method employed in the field of forensic science (Yokoyama and Uchimura, 2007).
It allows the identification and separation of a single individual from a group of individuals.
References Lindstedt BA, Heir E, Gjernes E and Kapperud G (2003): DNA fingerprinting of Salmonella enterica subsp. enterica serovar typhimurium with emphasis on phage type DT104 based on variable number of tandem repeat loci. J. Clin. Microbiol. 41(4):1469-79. Yokoyama E and Uchimura M (2007): Variable number of tandem repeats and pulsed-field gel electrophoresis cluster analysis of enterohemorrhagic Escherichia coli serovar O157 strains. J. Food. Prot. 70(11):2583-8.