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Amplification of Genomic cheek cell DNA through PCR method, and Hardy-Weinberg equation By Jamila Obsiye Texas A&M University Department of Biology Commerce, TX 75428

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Abstract: DNA, RNA, proteins, and enzymes all have in common a progressing perennial quest among scientists to understand their complex appliance, and functions. They are present in many organisms, and in diverse amounts bringing to question the advances that could be made in the health and science field if further knowledge was accumulated. To better understand the mechanism of molecular biology, this study focuses on DNA and the PCR method. With the use of the Polymerase chain reaction, DNA could now be extensively studied without requiring such a high level of skill. This means that a single trace amount of DNA could be amplified into a large amount showing the precise sequence. This discovery has made a huge impact, making this study one of the most open fields of biotechnology that aims to improve our society today and for the future. Keywords: DNA amplification; PCR; Hardy-Weinberg equation

Introduction DNA, or Deoxyribonucleic acid Is a critical structure within the nucleus of an organism that holds genetic information. The chemical structure of DNA has distinct sequences of nucleotides that form a Chromosome. These genes contain the necessary information to produce essential functional products like protein that are the building blocks of a healthy organism. The Genome is our hereditary code composed of DNA that regulates our physical and psychological behavior. In order to better understand ourselves as human beings, scientists strive to understand our molecular framework through DNA. In 1983, Kary Mullis developed a technique called polymerase chain reaction that drastically helped scientist study DNA effectively. Our lab group used the PCR method to magnify sample DNA from our cheek cells, and enhance the results so that we could categorize our genotypes. Once we went through the

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four stages of genetic fingerprinting, we then calculated our frequencies and applied the HardyWeinberg equation to test the deviation of our results so that we could find our hypothesis. We have two hypothesis; the first being that the frequencies of our lab class will not match the Hardy-Weinberg equilibrium compared to the USA population due to the three assumptions not being met. Our null hypothesis is that the genotype frequencies are determined by the random arrangements of alleles based on their frequency in the population. Methods and Materials To isolate genomic DNA we first had to obtain sample living cheek cells. A volunteer in our lab group rinsed a cup with 10 ml of .9% saline solution in their mouth for 30 seconds, and then transferred it into a micro test tube. We then centrifuged the micro test tube to clot the cheek cells, and then repeated this process until we concentrated the cells. Afterwards, we resuspended the cells and transferred them to the screwcap tube containing the InstaGene. Subsequently we collected our sample, incubated the tube at 56 degrees C for 10 minutes, followed by vortexing and incubating at 100C for 5 minutes. Finally we suspended the cell concentration and placed it in the centrifuge for a total of 5 minutes to obtain our DNA template. This DNA template then underwent PCR to specifically amplify a locus on human chromosome 16 so that we could read the Alu (unique base pair sequence) outcomes to determine the genotypes. The hypotheses we tested were the null hypothesis (H0) that proclaimed the genotype frequencies are randomly determined by the arrangement of alleles based on their frequency in the population, and that there would be no change. Our alternate hypothesis (Ha) was there would be a difference between the lab groups and the outcome of their genotypes. After the PCR amplification of the cheek cell DNA was completed, we then examined the electrophoresed product on agarose gel. There was a possibility of three

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outcomes: if both chromosomes contain Alu inserts, then each amplified PCR product would be 941 base pairs long and homozygous positive; if neither chromosome contained the insert, then each amplified PCR product would be 641 base pairs long and homozygous negative; if only one chromosome contained the insert, then there would be two bands, one with 641 base pairs, and another with 941 base pairs making the sample heterozygous. A Hardy-Weinberg equation was then used to compare, and test the deviation of our lab classes result with a similar sample taken from a large general united states population. These calculations made from the Hardy-Weinberg equation would also act as a base, or an ideal state for contrast. Since we were testing the frequencies of the genotypes, our lab group first had to find the 2 important variables in the equation. The P value and the Q value; respectively frequency of the dominant (+) and frequency of the recessive (-) genotype. After finding these frequencies by dividing the amount by total, we then calculated our Q 2 and P2 values by squaring the P and Q values. For our heterozygous genotype, we multiplied our P and Q value by the coefficient 2. If the study met the hardy Weinberg equilibrium then the initial observed genotypes would equal the expected frequencies from the Hardy-Weinberg calculation. We then also calculated our chi-square value to specifically test this deviation so that we could accept or reject our hypotheses. After we subtracted 1 from our number of outcomes to get our degree of freedom, we then proceeded to subtract our observed frequencies from expected. Subsequently, we squared that number and divided it by the number of the expected for that outcome. Our basis of comparison so that we could reject or accept our hypotheses was based on the significance of the the value underneath .05. For the df value of 2, our expected frequency was 5.99. Anything less than that value would basically accept our null hypothesis.

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Results Two lab groups were Homozygous dominant, 2 were Homozygous recessive, and three were heterozygous dominant. For the Hardy-Weinberg equation the disparity between observed Genotypic Frequencies for the class in (p=.71, q=.29) and the observed Genotypic Frequencies for USA (p=.8, q=.2) though not too great, was solid (table 1, table 3). The deviation between the observed values for the class, and the calculated (p 2=.5041, q2=.0841, 2pq=.4118) also was notable. The USA population calculated frequencies (p2=.64, q2=.04, 2pq=.64) likewise was not similar to its observed Genotypic Frequencies. The Chi square value had these numbers corresponding to the outcomes: Homozygous dominant expected-.71, observed-.29; Heterozygous dominant expected-.4118, heterozygous observed-.42, and homozygous recessive expected-.29, observed-.29. The x2 value is .25016, and the degree of freedom is 2. Discussion The progression of modern technology to compliment the field of sciences has resulted in a broad study of BioInformatics. This coupling has led to many advances, and discoveries whether in environmental, cell, or molecular biology. The importance of this dyad has resulted in many of the breakthrough discoveries one can witness today; Whether it be the Human Genome project attempting to chart the migration of human beings, or to find the perpetrator to the crime scene. All of these relate in that they utilize the pairing of science and technology to solve their problems. The PCR method has allowed us to magnify our cheek cell DNA for further study on the organization, and traits within this microscopic structure. Through the test we did with the Hardy-Weinberg equation, and the Chi-square test, the deviation of the

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observed frequencies helped us decide to reject our null hypothesis, and accept our alternate hypothesis. More specifically, in the chi square test, our results were greater than 5% leading us to this conclusion. We also did not accept the Hardy-Weinberg equilibrium because our control did not meet the three assumptions of the model. Our study group did not a large population, and there was no way to determine if the mating was random, or if there was excessive mutation between the alleles. Our basis of comparison was the USA population, which also did not meet the Hardy-Weinberg equilibrium. Our conclusion is that neither of these tested groups could meet the equilibrium because they were not in a fully controlled environment. The ideal basis for hardy-Weinberg though did allow us to see that evolution was possible, since our genotype frequencies were completely different. Our actual class data was not drasitally from from the calculated genotypic and allelic frequencies though. There was no expectation for the results of our lab class to match the results of the USA population since there were differences like the population, and location. There also was a factor of other possibilities since our environment was not perfectly controlled. While we were doing the experiment, we did not precisely pipette the DNA into the wells. Human error should be taken into consideration, as well as variation from exact timing.

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Citations
Abd-Elsalam, Kamel A. Bioinformatic Tools and Guideline for PCR Primer Design. Rep. Molecular Markers Lab., Plant Pathology Research Institute, Agricultural Research Center, Orman 12619, Giza, Egyp, 28 Apr. 2003. Web. Nov.-Dec. 2012. Lab Manual 10, DNA fingerprinting DNA EXTRACTION VIRTUAL LAB. Computer software. DNA Extraction Virtual Lab. The University of Utah,Supported by a Science Education Partnership Award (SEPA) [No. 1 R25 RR16291-01] from the National Center for Research Resources, n.d. Web. 27 Nov. 2012. <http://learn.genetics.utah.edu/content/labs/extraction/>. S A Bustin, V Benes1, T Nolan2 and M W Pfaffl3. "Journal of Molecular Endocrinology."Quantitative Realtime RT-PCR a Perspective. The Society for Endricronology, 2012. Web. 27 Nov. 2012. <http://jme.endocrinology-journals.org/content/34/3/597.full>. Copyright 2004-2012, Prof B. Jayaram & Co-workers. "What Is Gene." What Is Gene ?N.p., 2004,2012. Web. 27 Nov. 2012. <http://www.scfbio-iitd.res.in/tutorial/gene.html>.