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Unformatted text preview: Exercise 6. Analysis of the Alu Insertion and Cell Division 79 EXERCISE 6 PCR AMPLIFICATION OF AN ALU POLYMORPHISM, AND GENETIC TRANSFORMATION "In the field of observation chance favors only the prepared mind." Louis Pasteur in a lecture at the University of Lille 1854" * THE POLYMERASE CHAIN REACTION * THE MOBILE GENETIC ELEMENT ALU IN THE HUMAN GENOME * TRANSFORMATION OF BACTERIA WITH A PLASMID Equipment and supplies needed PCR of Alu element 15 ml tube with 10ml sterile saline paper cups microcentrifuge tubes microcentrifuge clinical centrifuge and timer plastic transfer pipettes ready to go PCR tubes P-20 pipetmen and tips thermocycler vortex mixer Chelex (10% in 50mM Tris pH 10.5) heat block 100C sterile transfer pipettes heat blocks 42C ice container PCR computer animation primer mix Bacterial Transformation competent E. Coli plates GFP plasmids transformation buffer (CaCl2) LB solution Vortex mixer LB media The purpose of this exercise is to amplify specific regions of human DNA using the powerful technique known as PCR (the polymerase chain reaction), and to introduce into bacterial cells a plasmid containing DNA that codes for the green fluorescent protein Exercise 6. PCR Amplification of TPA 25 and Genetic Transformation 80 PART I. The Polymerase Chain Reaction Today you will use a very powerful technology, the polymerase chain reaction, to examine a region of your own DNA. What is the polymerase chain reaction and why is it so important? In 1993, the Nobel Prize for Chemistry was awarded to Kary Mullis for the development of a technique called the polymerase chain reaction. PCR is an extremely clever method to amplify (make many copies of) DNA fragments. All of the components of PCR have been known for some time, but the genius of Dr. Mullis was the assembly of the components into a single procedure. The critical component of the procedure is the DNA polymerase produced by a thermophilic organism. Thermophilic organisms (bacteria and eukaryotes) grow at high temperatures, often approaching or exceeding 100 o C! Obviously their DNA and enzymes, such as their DNA polymerases, function well at these high temperatures since they replicate their DNA prior to each cell division. The DNA of most organisms, however, denatures at high temperatures because the hydrogen bonds between the bases break. The strands of the double helices detach and separate. In the polymerase chain reaction, the thermostable DNA polymerase, nucleotide building blocks, and DNA are heated in a solution containing high concentrations of small primers which are complementary to specific sequences on the DNA. As the reaction mixture is cooled, the primers hybridize to the DNA before the long DNA strands reanneal to each other because the primers are small and present in high numbers....
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- Spring '10