Lec39n - Lecture 39 DNA Technology, Part 2. PCR, RFLPs,...

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Lecture 39 DNA Technology, Part 2. Campbell, Chapter 20; 6 th Ed. pages 382-386, 393 7 th Ed. 391-398. 8 th Ed. 403-409 PCR. The polymerase chain reaction (PCR) is used to amplify, or make many copies of, a given DNA sequence, in vitro (that is, in a test tube, without cells). This contrasts with the gene cloning we discussed last time, which is done by introducing genes into a plasmid, putting the recombinant plasmid in bacteria, and letting bacteria replicate the plasmid. In addition to the fact that we don’t need bacteria, the advantages of PCR are the following: it’s fast, it needs very little starting material, and it’s not limited by a need for restriction enzyme recognition sites. Any piece of DNA can be amplified. For PCR, we need a DNA template, a special heat-stable DNA polymerase (that can withstand the heat required to denature the DNA strands), dNTPs, and TWO DNA primers that are complementary to short sequences on OPPOSITE STRANDS of the targeted sequence. The 5’ end of each primer marks one end of the targeted sequence (see Fig. 1). The primers are included in huge excess over the template. The method is to repeat the following 3-step cycle 25 or 30 times (Follow along in Campbell, Fig. 20.7, 6 th th ; 20.8, 8 th Ed.). Step 1; heat the DNA to denature (i.e., to separate the 2 strands). Step 2; cool to allow complementary base pairing of the template and the primers. (Primers bind template more often than template strands bind each other, because they’re present in excess). Step 3; extend, allowing DNA synthesis to occur from the primers, using the template DNA as a guide. During the first cycle, each primer binds a strand of the template, and is extended into a new long strand. After awhile, we stop the extension reaction and start Cycle 2, by heating again. When we do this, the new strand and the template strand come apart from each other. (This means that the new long strand made in Cycle 1 has an arbitrary length: it ends wherever DNA polymerase happened to be when we heated to start Cycle 2.) We now cool to let new primers bind again. At this point, each primer has 2 choices. It can either bind to the complementary original template strand. If it does this, during extension, it will make another strand, just as in Cycle 1. However, instead, the primer might bind to the new strand that was made in Cycle 1 starting with the OTHER primer. (This new strand has the same sequence as the template strand that our primer recognizes). Note that this new strand is not very long. For this reason, if a primer binds to it, and DNA polymerase uses it as a template during the extension reaction of Cycle 2, it will soon reach the end and fall off. This strand (made in Cycle 2) will be short – only as long as the distance between the 2 primers. In other words, it’s the desired fragment! Each new strand that’s made in the PCR reaction can serve as a template in the next cycle.
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Lec39n - Lecture 39 DNA Technology, Part 2. PCR, RFLPs,...

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