Abstract
A polymerase chain reaction also known as PCR refers to a technique that is used in the amplification of a DNA fragment making huge numbers of the original fragment. The process uses an enzyme known as DNA polymerase to multiply the DNA fragment. Other components that are necessary for a PCR reaction are the DNA template that is provided by the sample being multiplied, the primers, and nucleotides. The experiment aimed to examine a number of human DNA polymorphisms, the PV92 Alu polymorphism and the mitochondrial control region using human DNA. This was achieved by isolating the mitochondrial DNA from the mouth cells and performing PCR amplification to increase the DNA sample. The samples were run through gel electrophoresis to establish polymorphism. The experiment successfully illustrated the PV92 Alu polymorphism in a number of individuals.
Introduction
A polymerase chain reaction also known as PCR refers to a technique that is used in the amplification of a DNA fragment making huge numbers of the original fragment (Bartlett and Stirling). The process uses an enzyme known as DNA polymerase to multiply the DNA fragment. Other components that are necessary for a PCR reaction are the DNA template that is provided by the sample being multiplied, the primers, and nucleotides (Sambrook, Fritsch and Maniatis).
In the PCR process, a set of four different steps are repeated for a number of 20 to 40 cycles. The steps start with the initiation step, denaturation, annealing and elongation steps. The initiation step involves heating the reaction mixture to a temperature greater than 90°C. Once the process is initiated, the process undergoes a denaturation step where the two stranded DNA strands are separated after being heated at 94–98 °C for about 30 seconds. This results in the formation of single-stranded DNA molecules (Gelder and Russell). The primer then anneals to the single stranded DNA molecules at a specific site at a temperature of between 50 and 65 °C depending on the composition of the nucleotides in the primer. Once the primers have annealed, the primer is elongated by adding nucleotides to the primer. This occurs mainly at about 75°C and the addition of the nucleotides is enhanced by the DNA polymerase enzyme (Sambrook, Fritsch and Maniatis).
Occurrence of two or more different phenotypes or genotypes in the same species making up population is referred to as polymorphism. These variations are hereditable, and their presence can be used in the determination of parentage or ancestry, in forensic research and other purposes. Length polymorphisms can be displayed by PCR followed by agarose gel electrophoresis. The separated polymorphisms sequences can then be analyzed by PCR followed by DNA sequencing.
This experiment aimed to examine a number of human DNA polymorphisms, the PV92 Alu polymorphism and the mitochondrial control region using human DNA.
Methods
Alu insertion polymorphism
This experiment aimed to examine PV92, a locus on chromosome 16 in which a H. sapiens-specific Alu insertion may or may not be present.
Part I: DNA Isolation by Saline Mouthwash
Into a 15 ml polypropylene culture tube, 10 ml 0.9% saline solution was prepared by dissolving 0.9 g NaCl in 100 ml of distilled water, and into a 1.5 ml polypropylene tube, 100 µl of 10% Chelex® suspension was prepared. Before starting the lab, the mouth was rinsed with water at the water fountain, and 10 ml of the saline solution (0.9% NaCl) was poured into the mouth. The solution was swished vigorously in both cheeks for 1 minute and the saline solution expelled into a paper cup. The solution was swirled gently in the dish and 1ml of the liquid transferred to a 1.5 ml capped microcentrifuge tube. The sample tubes were placed in a balanced configuration in a microcentrifuge and spun for 1.5 minutes. The tube was opened gently, and the supernatant poured off into the paper cup. The cells were resuspended in saline by pipetting in and out using a P-200 pipette. From the mixture, 30 µl of cell suspension was withdrawn and added to the tube containing 100 µl of Chelex. The tube was tightly capped and mixed through vortexing. The cell sample was boiled for 10 minutes using the thermal cycler at 99°C and then cooled briefly on ice. The content was mixed, and the samples placed in a balanced configuration using a microcentrifuge, and spun for 30 seconds. Into a clean 1.5 ml tube, 30 µl of supernatant that contained the DNA was transferred and the tube stored on ice.
Part II: DNA Amplification by PCR
Using a P-200 micropipette with a fresh tip, 22.5 µl of the appropriate primer/loading buffer mix was added to a PCR tube containing a Ready-To-Go PCR Bead. The tube was tapped with a finger to dissolve the bead. Using a fresh tip, 2.5 µl of the human DNA extracted were added to the reaction tube and the tube tapped to mix the content. The reagents were pooled by pulsing in a microcentrifuge and the cap labeled with a number. All the samples were stored on ice and the thermal cycler programmed as shown in Table 1 below.
Part III: DNA Analysis by Gel Electrophoresis
Using a micropipettor with a fresh tip, 15µl of the PCR sample/loading dye mixtures were added into the assigned wells of a 1.5% agarose gel. Into one lane of gel, 5 µl of the pBR322-BstNI size markers were loaded into one lane of gel. The gel was run at 130 volts for 30 minutes to enable adequate separation.
Analysis of Human DNA Alu polymorphism
Part I: DNA Isolation by Saline Mouthwash
The DNA sample isolated in the previously was used for the following procedures.
Part II: DNA Amplification by PCR
Using a P-200 micropipette with a fresh tip, 22.5 µl of the appropriate primer/loading buffer mix was added to a PCR tube containing a Ready-To-Go PCR Bead. The tube was tapped with a finger to dissolve the bead. Using a fresh tip, 2.5 µl of the human DNA extracted were added to the reaction tube and the tube tapped to mix the content. The reagents were pooled by pulsing in a microcentrifuge and the caps labeled with a number. All the samples were stored on ice and the thermal cycler programmed as shown in Table 2 below.
Part III: DNA Analysis by Gel Electrophoresis
Using a micropipettor with a fresh tip, 15µl of the PCR sample/loading dye mixtures were added into the assigned wells of a 1.5% agarose gel. Into one lane of gel, 5 µl of the pBR322-BstNI size markers were loaded into one lane of gel. The gel was run at 130 volts for 30 minutes to enable adequate separation.
Results
The gel electrophoresis results for the DNA samples that were isolated from the mouth were as shown in Figure 1 below. The presence of the mitochondrial DNA was seen in most of the samples that were tested with others having a faint band and others showing no band at all.
Figure 1: The gel electrophoresis results for the analysis of the mitochondrial DNA
In the determination of the Alu insertion polymorphism, the gel electrophoresis results for the DNA samples that were amplified using PV92 primers were as shown in Figure 2 below. The presence of the two variations of Alu element was seen in some individuals (1, 3, 4, 7, 15, 18, 20 and 23), only one variation in others (2, 10 and 13) while others had no Alu element at all (5, 6, 8, 9, 11, 12, 14, 16, 17, 19, 21, 22 and 24).
Figure 2: The gel electrophoresis results for the analysis of the mitochondrial DNA
The gel for the analysis of d1s80 polymorphism indicated that there was polymorphism in some individuals where some had the two different alleles others had either of the two alleles and others had no allele at all (Figure 3).
Figure 3: The gel electrophoresis results for the analysis of the mitochondrial DNA for the D1S80 polymorphism
Discussion
The mitochondrial DNA has a number of differences between individuals of the same species resulting in variations. The variations occur at the control region and result from the presence of Single Nucleotide Polymorphisms (SNPs), single nucleotide insertions or substitutions or deletions. In the control region, there are two regions that are located within the 1.1 Kb fragment showing multiple variations in different individuals belonging to the same population. The two variable regions that are located in the control region are the hypervariable region 1, also known as HV1 and the hypervariable region 2, also known as the HV2. The sequence information that is covered for HV1 region ranges from p16,024 to p16,365. The sequence information that is covered for HV2 region covered from p73 to p340.
In the current experiment, the amplified region by PCR corresponded to the HV1 as shown in Figure 1. Most of the samples tested were successful in the showing the presence of the mitochondrial DNA in the samples collected. The absence of a band in other samples may have resulted in errors, in the isolation of the DNA content from the cheek cells or failure in the amplification of the DNA template during PCR process. In the samples that gave a band in the sample, the bands were all at the same level, and this was an indication of successful targeting of the desired DNA fragment. The primer used was thus able to anneal to the DNA template followed by the other steps of PCR successfully.
The PV92 genetic system consists of only two alleles. The alleles may be present in either the chromosomes inherited from the mother and the father or in only one of the two chromosomes. Presence of the PV92 in one chromosome resulted in only a single band, in a sample (+/+). This was the case for the 8 of the samples that were tested indicating that the individuals inherited both the chromosomes having the PV92 element. The presence of the PV92 in the two chromosomes resulted in two bands, in the sample (+/-). This was the case for the 3 of the samples that were tested indicating that the individuals inherited only one of the chromosomes having the PV92 element. Lack of the PV92 in either of the chromosomes resulted in the absence of the band in the sample (-/-). This was the case for the 13 of the samples that were tested indicating that the individuals inherited both the chromosomes that had no PV92 element. The results also showed the presence of D1S80 polymorphism. Some of the individuals had different alleles in the two chromosomes while others had the similar chromosomes in the two chromosomes. There were others who had no band present in the gel. This indicated either the reaction had errors that prevented the experiment to succeed or no allele in the two chromosomes.
In conclusion, the experiment successfully examined a number of human DNA polymorphisms, the PV92 Alu polymorphism and the mitochondrial control region using human DNA. The experiment may be taken further by determining the sequence for the different fragments that were observed.
Works Cited
Bartlett, J. M. and D. Stirling. "A short history of the polymerase chain reaction." PCR protocols 226 (2003): 3-6.
Gelder, Elma Kim and N. Van Russell. Duane's Ophthalmology. Philadelphia: Lippincott Williams & Wilkins, 2007.
Sambrook, J., E. F. Fritsch and T. Maniatis. Molecular Cloning: A laboratory manual. 3rd. New York: Cold Spring Harbor Laboratory Press, 2001.
