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Analyzing the PV92 locus on chromosome 16 for the Alu insertion through the polymerase chain reaction and

agarose gel electrophoresis

Johansen Pico Principles of Forensic Science, Northwest Career and Technical Academy

ABSTRACT The experiment explores the significance of SINEs, also known as short interspeed elements, in the human genome. The SINE that we targeted was the Alu sequence on the PV92 locus on chromosome 16. This is possible through the polymerase chain reaction, where DNA samples undergo thermal cycling, and agarose gel electrophoresis, where amplified samples of DNA are able to run through a gel for visualization. After the experiment was conducted, we determined the zygosity of the Alu sequence with DNA samples from lab group members. We were able to display DNA strands that were homozygous recessive, homozygous dominant, and heterozygous for the Alu sequence. Through our findings, we were able to determine the significance of SINEs regarding genomic differentiation and human identification.

INTRODUCTION Deoxyribonucleic acid, commonly shortened to DNA, is hereditary material that is found in each of our cells, specifically the nuclei. DNA derives from four chemical bases, adenine, guanine, cytosine, and thymine. These bases pair with each other: A with T and C with G. The human genome alone consists of 3 billion of these base pairs, also shortened to bp, 95 percent of which is non-coding DNA. The arrangement of these base pairs depends on what it is expressing (Mandal, 2012).

DNA is constantly being replicated in cells, which is a prerequisite to cell division. The process involves the production of two identical replicas from the original DNA strand, which is possible because of cellular proofreading. DNA polymerases, a group of enzymes, perform all types of replication (Berg, 2002). Polymerases cannot initiate synthesis of new strands, but they extend existing DNA or RNA strands paired with a template strand. Synthesis begins with a short fragment of RNA, termed a primer. Strands of DNA have directionality: the left end of the sequence being the 5 (pronounced 5-prime) end, while the right end of the sequence is the 3 (pronounced 3prime) end, and the opposite holds true for the corresponding strand (Alberts, 2002). This has an effect with replication, as polymerases are only able to extend in one direction, which is through the addition of nucleotides to the 3 end of a strand (Alberts , 2002).

Fig. 1. Extension of DNA strand with DNA polymerase. Adapted from Lizabeth A. Allison, 2007, Fundamental Molecular Biology, p. 112. The figure displays extension through the addition of nucleoside triphosphate on the 3 end of the strand.

DNA polymerases and primers can be used to perform in-vitro replication. Isolated polymerases and artificial primers can be used to emulate DNA synthesis at known sequences in template DNA molecules (Alberts, 2002). The polymerase chain reaction, shortened to PCR, is a lab technique that utilizes in vitro replication in order to amplify a desired DNA fragment, creating anywhere from thousands to millions of copies of a desired sequence (Bartlett, 2003). PCR depends on thermal cycling, where the sample is repeatedly heated and cooled for DNA melting and enzymatic replication of the DNA (Carr, 2003). A thermal cycler is commonly used in labs for this process (Weier, 1988). The first step of thermal cycling is where the DNA is heated to 94-96 degrees Celsius for several minutes, where the sample is denatured into single strands. Next, the sample is cooled to 50-65 degrees Celsius for several minutes, where the primers hybridize, or anneal, to corresponding sequences on either side of the target sequence. Lastly, the sample is heated to 72 degrees Celsius where the DNA polymerase binds and extends complementary DNA strands from each primer. The cycle repeats thirty times, and the process yields approximately one billion copies of the targeted sample (Bloom, 2008).

Fig. 2. Illustration of classical PCR. Adapted from Juan G. Santiago, 2009, On-chip device for isothermal, chemical cycling polymerase chain reaction. Displays double-stranded DNA undergoes

three cycles where it denatures, anneals primers, and is extended by polymerase. With proper conditions, the number of copies double after each cycle.

In forensic science, PCR can be used to identify genetic fingerprints through different loci in the human genome. Certain DNA sequences that are repeated, e.g. GTGTGT, are random for every individual, and as a consequence, makes identification through forensic techniques effective (Alberts, 2004). The visualization of base pairs after performing PCR is possible through gel electrophoresis, which utilizes a gel as a medium for DNA samples to move and separate (Bianchi, 2010). In this experiment, we will be targeting the Alu sequence in the PV92 locus on chromosome 16. Alu elements are short interspeed elements, commonly referred to as SINEs. SINEs are repetitive sequences that make up approximately 13% of the human genome, and they are genetically neutral, which means that they do not code for any specific function (Singer, 1982). This sequence is 300 bp long, and it can be found almost 500,000 times throughout the human genome. Since the sequence is considered dimorphic, the insertion may be found in certain individuals, and others may not. Some individuals may have the Alu sequence on one copy of the 16 th chromosome, while others may have it on both copies, and some may not have the sequence on either chromosome (Batzer, 1991). Determining whether an individual has the Alu sequence is possible through comparing DNA samples with control samples through PCR and agarose gel electrophoresis. After this experiment, we will be able to self-identify the presence of the Alu insertion in either one, two, or no alleles on chromosome 16, which in turn can lead to various real-world applications with genomic identification.

MATERIALS AND METHODS For this experiment, we first collected 200 microliters of Instagene was obtained for each DNA sample. Two hairs with root and sheath were placed into individual tubes. To begin the polymerase chain reaction, the tubes were heated at 56 degrees celsius for 5 minutes, vortexed gently, and then heated at the same temperature for 5 more minutes. After ten minutes, the tubes were vortexed again. After five minutes of the previous cycle, the tubes were heated at 100 degrees celsius. Afterwards, we removed the tubes from heat, and centrifuged them for 5 minutes. We drew twenty microliters of the DNA, drawing from the supernatant, carefully not drawing from the pellet created from centrifuging, and then added it to a labeled PCR tube containing 20 microliters of master mix. To prepare for our agarose gel electrophoresis, a 1% agarose gel was created with 50 mL of 1X TAE buffer and 0.50g of agarose. We initially started with 50X TAE buffer, but we were able to dilute the sample to 1X by adding 5 mL of the 50X TAE buffer and 995 mL of water to a 1L bottle. The .05g of agarose powder was added to the Erlenmeyer flask of 50 mL 1X TAE buffer. The flask was heated at 10 second intervals until contents have been dissolved. After cooled, one drop of Ethidium bromide was added to the flask, and the solution was poured into the electrophoresis box. Two 10 toothed combs were placed in the box to provide each group member a well for their DNA samples was inserted into the gel. After solidification, TAE buffer was poured into the box, which allows electricity to flow through the box in order for our gels to run. After the PCR tubes were placed in the thermal cycler and were amplified through the process of PCR, 10 microliters of PV92 XC loading dye was added to the

tube and mixed gently in order to be visualized after we ran our samples through the gel. A clean tip was used to load each sample into their designated wells in the agarose gel. After loading, the electrophoresis box was connected to the power supply and the samples were run at 100 V for 30 minutes. After electrophoresis, the gel would be able to be visualized under a UV-transilluminator.

RESULTS After the completion of this experimental procedure, we determined the presence of the Alu sequence on each group member s chromosome 16. Figure 3 displays a photo of our agarose gel after running for thirty minutes.

Fig. 3. Agarose gel after completion of electrophoresis displayed through a UV-transilluminator. Lane samples in order from left to right: Ladder, Homozygous dominant for Alu, Heterozygous for Alu,

Homozygous recessive for Alu, John Belbis, Cole Lloyd-Leakos, Jake Pepito, Johansen Pico, Tricia Ramos, and Raymond Vinuya.

By comparing our group members samples to the control samples in lanes 2-4, we are able to determine each group member s presence, or absence, of the Alu sequence on their respective chromosome 16. Group members Jake Pepito, Tricia Ramos, and Johansen Pico in lanes 7-9 tested homozygous dominant for the Alu sequence. Cole Lloyd-Leakos and Raymond Vinuya in lanes 6 and 10, respectively, tested homozygous recessive for the Alu sequence. One group member, John Belbis, in lane 5, had ambiguity with results because his band was thinner than any of the control groups, but it does seem plausible that he may test for homozygous dominant.

DISCUSSION Through our experiment, we were able to display the dimorphic trait of the Alu sequence in the human genome. By analyzing chromosome 16 using hair samples, we were able to determine the presence, or absence, of the Alu sequence in each group members genome. Being homozygous dominant for the trait meant that both pairs of chromosomes had the trait, heterozygous meant that only one pair had the sequence, and homozygous recessive meant that no chromosome had the sequence at all. Our experiment was able to show that individuals are able to be distinguished through the presence of the Alu sequence. Although the Alu sequence is not known to code for any specific human function as of yet, the underlying significance of the results does not lie with this fact. Being able to distinguish individuals through this genomic sequence alone is enough to identify

individuals on the genomic level. Different lab groups that also participated in this experiment may have tested samples from family members, which can display whether the parent is a biological parent, or not. This is an example of how important SINEs play a function in genomic identification because if an offspring is homozygous dominant for the Alu sequence, and a parent is homozygous recessive, the parent is highly unlikely to be of biological relation to the offspring. Along with paternity testing, SINEs can also play a role in determining the role of suspects at a crime scene. Being able to establish and determine identity with genetically neutral DNA is a significant step in furthering the validity of forensic science. One challenge that may come across with using the PV92 locus is that some samples may not visualize correctly the first time, which can be portrayed in the sample on lane 5 in Figure 3. This particular sample has a thinner band than our control samples, and as a result, skews the validity and results of this test through the ambiguity of the sample. If this were to happen in a case scenario, the confidence level of the test significantly drops, and would require further time and resource to re-test the individual for the Alu sequence. However, this may have just been a case where the sample may not have had enough DNA to amplify during the PCR process, so the band appeared to be significantly thinner than the rest. In a future study, I would study the trend of the appearance of the Alu sequence on the PV92 loci with different human characteristics, specifically gender and ethnicity, to determine whether certain genders or ethnicities are more likely to share a trend in the appearance of the Alu sequence, or not. The study could prove to be significant in identification if the results determine whether certain genotypes are more likely to have

the sequence, or not. Results could then be extrapolated to different populations, and can be used to reduce the amount of suspects in certain criminal cases involving DNA.

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