1 a complete cycle of pcr the target sequence the

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Unformatted text preview: highly dependent on temperature. In this lab exercise, a 60°C annealing temperature is optimum for primer binding. During the extension step, the job of Taq DNA polymerase is to add nucleotides (A, T, G, and C) one at a time to the primer to create a complimentary copy of the DNA template. During polymerization the reaction temperature is 72°C, the temperature that produces optimal Taq polymerase activity. The three steps of denaturation, annealing, and extension form one “cycle” of PCR. A complete PCR amplification undergoes 40 cycles. The entire 40 cycle reaction is carried out in a test tube that has been placed into a thermal cycler. The thermal cycler contains an aluminum block that holds the samples and can be rapidly 1 Bio 05LA – Fall Quarter 2012 Lab 8 heated and cooled across broad temperature differences. The rapid heating and cooling of this thermal block is known as temperature cycling or thermal cycling. 3' 5' 5' 3' Denature strands at 94°C 5' 3' 3' 5' Anneal primers at 60°C (Taq polymerase recognizes 3' ends of primers) 3' 5' Primer 3' 5' Taq polymerase 3' 5' Primer 5' 3' Extend at 72°C (Synthesize new strand) 5' 3' 3' 5' 5' 3' 5' 3' Repeat cycle 40 times Fig. 1. A complete cycle of PCR. The Target Sequence The human genome contains small, repetitive DNA elements or sequences that have become randomly inserted into it over millions of years. One such repetitive element is called the “Alu sequence”. T hi s i s a D N A s equenc e a bou t 3 0 0 b as e pairs lon g t ha t i s repeated almost 500,000 times throughout the human genome. The origin and function of these repeated sequences is not yet known. In this laboratory activity you will look at an Alu element in the PV92 region of chromosome 16. This particular Alu element is dimorphic, meaning that the element is present in some individuals and not others. Some people have the insert in one copy of chromosome 16 (one allele), others may have the insert in both copies of chromosome 16 (two alleles), while some may not have the insert on either copy of the chromosome (Figure 2). The primers in this kit are designed to bracket a sequence within the PV92 region that is 641 base pairs long if the intron does not contain the Alu insertion or 941 base pairs long if Alu is present. This increase in size is due to the 300 base pair sequence contributed by the Alu insert. 2 Bio 05LA – Fall Quarter 2012 Lab 8 When your PCR products are electrophoresed on an agarose gel, three distinct outcomes are possible. If both chromosomes contain Alu inserts, each amplified PCR product will be 941 base pairs long. On a gel they will migrate at the same speed so there will be one band that corresponds to 941 base pairs. If neither chromosome contains the insert, each amplified PCR product will be 641 base pairs and they will migrate as one band that corresponds to 641 base pairs. If there is an Alu insert on one chromosome but not the other, there will be one PCR product of 641 base pairs and one of 941 base pairs. The gel will reveal two bands for such a sample. Fig. 2. The presence or absence of the Alu insert within the PV92 region of chromosome 16. PV92 Genotype DNA Size of PCR Products ALU Homozygous (+/+) 941 base pairs Homozygous (–/–) ALU 641 base pairs ALU Heterozygous (+/–) 1 2 3 4 5 941 and 641 base pairs 6 7 8 (bp) 1,000 700 500 200 100 Fig. 3. Electrophoretic separation of DNA bands based on size. EZ Load DNA molecular mass ruler, which contains 1,000 bp, 700 bp, 500 bp, 200 bp, and 100 bp fragments (lane 1); two homozygous (+/+) individuals with 941 bp fragments (lanes 2, 6); three homozygous (–/–) individuals with 641 bp fragments (lanes 3, 5, and 8), and two heterozygous (+/–) individuals with 941 and 641 bp fragments (lanes 4 and 7). 3 Bio 05LA – Fall Quarter 2012 Lab 8 Electrophoresis separates DNA fragments according to their relative sizes. DNA fragments are loaded into an agarose gel slab, which is placed into a chamber filled with a conductive buffer solution. A direct current is passed between wire electrodes at each end of the chamber. DNA fragments are negatively charged, and when placed in an electric field will be drawn toward the positive pole and repelled by the negative pole. The matrix of the agarose gel acts as a molecular sieve through which smaller DNA fragments can move more easily than larger ones. Over a period of time, smaller fragments will travel farther than larger ones. Fragments of the same size stay together and migrate in what appears as a single “band” of DNA in the gel. In the sample gel above (Figure 3), PCR-amplified bands of 941 bp and 641 bp are separated based on their sizes. Lab Period 2 – Separation and Visualization of PCR Products The PCR products you generated last week will be separated by gel electrophoresis. After for loading samples into the wells of an agarose gel (A). The lid is then placed on the apparatus and the power cords are plugged into the power supply (B). Separation of the fragments will occur as the current flows through the gel. Note that the cords are color coordinated. The red cord represents the positive end of the field and the DNA will migrate toward that end of the gel (the dye front will be your visual indicator). After about 40 minutes at 120V the electrophoresis will be stopped and the gel transferred to a tray containing a concentrated staining solution (C). The DNA in the gel will bind the dye allowing visualization of the bands. A C B Can you determine whether you are homozygous (+/+), homozygous (-/-) or heterozygous? Genotype determined ____________________________________ *The introductory information and protocol has been adapted from “Biotechnology Explorer™- Chromosome 16: PV92 PCR Informatics Kit Manual which is a product of Bio-Rad. Duplication of any part of this document is permitted for classroom use only 4...
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This note was uploaded on 08/27/2013 for the course BIO BIOL05LA taught by Professor Abbottl during the Fall '12 term at UC Riverside.

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