The Polymerase Chain Reaction and Determination of Alu Population Freqencies
April 8, 2010
Cheek cells were extrapolated and used in PCR amplification and electrophoresis of the amplified samples to determine the presence or absence of the dimorphic Alu sequence in a class population. A bioinformatic allele server was then employed to calculate genotypic and allelic frequencies of the Alu element in the class population. The Hardy-Weinberg equation was also applied to individual data to produce class data for allelic and genotypic frequencies. Analysis of results and comparison to a larger population showed that those homozygous positive and possessing the sequence are in the smallest genotypic class. In smaller populations, those homozygous negative and not possessing the sequence were the largest genotypic classes, and in larger populations frequencies of heterozygotes and homozygous negative individuals were similar. Introduction
Prior to 1983, study of the human genome through biotechnology was limited only to the study of proteins and this study required such high expertise that very few scientists could conduct it. As studies continued, geneticists began to study not just the function of the enzymes that helped to make the proteins, but also how these enzymes themselves are made. This lead to a better understanding of the inner workings of organisms and how and why they function in the manner that they do. Today, geneticists study the enzymes, proteins, and nucleic acids to get a complete picture of the biological processes of organisms. In 1983, however, a new technique created by Kary Mullis called the Polymerase Chain Reaction (PCR) changed genetic research for the better for years to come. PCR is most commonly used to amplify a select region or DNA sequence from an entire genome. In PCR, large amounts of DNA are produced in vitro from very small trace amounts. DNA can be obtained from several different types of organic matter including blood and hair follicles. PCR is also used as a “cutting and pasting” mechanism to create recombinant DNA for study (Mullis 1994). The purpose of this experiment is to amplify a particular DNA sequence containing the dimorphic Alu insertion element to determine its presence or absence. Analysis of the Alu element, a non-coding sequence found on the PV92 region of chromosome 16, will be used to estimate its frequency in the class population as a means of measuring genetic variation among individuals.
Materials and Methods
The procedure followed the experimental design described in Laboratory Exercises in Genetics, by Dr. Melvin Beck. The experiment began with the preparation of the cheek cell DNA template using InstaGene matrix and a 0.9% saline solution to collect cells by swishing in the mouth. After the cell pellet was collected and resuspended in the test tube, the solution underwent a ten minute preincubation period at 56 degrees Celsius. The cells were then placed in a 100 degrees Celsius boiling water bath to rupture the cells and release the DNA contained within them. PCR Amplification was performed during the following class period. Standard procedure for PCR Amplification was conducted including the three steps of denaturation at 94 degrees Celsius, in which the double-stranded DNA was separated via melting into single-stranded DNA, annealing at 60 degrees Celsius, in which the oligonucelotide primers located and binded to their complementary template DNA strands, and extension at 72 degrees Celcius, in which the Taq polymerase, a thermophilic bacterium that can withstand the high level of heat, added the correct nucleotides to synthesize a new strand of DNA (Erlich 1989). This three-step process was repeated for 40 cycles. Gel electrophoresis of the PCR samples was then conducted using a 0.8% agarose gel matrix to determine the presence or absence of the Alu insert in the PV92 region of...
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