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Unformatted text preview: 3.29.07 Last step of cloning, DNA sequencing because you have to know what it is youve cloned, a confirmation step Back to the first step o Easiest way to do it is PCR Start with a template You selectively replicate just the target sequence you want to clone You target to a specific sequence with two primers, the two primers define the two ends of the target gene One primer is complimentary to the beginning of the sequence you want and the other primer is complimentary to the end of the sequence that you want The primers are single stranded DNA You have to know what the sequence is going to be and decide what those sequences are on either side, and how do you know what it is if you are isolating a gene youve never sequenced before The hardest part of PCR is deciding what primers to use because you have to know a lot about the sequence your trying to isolate Say you are able to know what the primers should be, how do you make the single stranded DNA? DNA synthesizer machine or write down the sequence and order them as oligos Need DNTPs as well because its replication in a way Just regular DNTPs, nothing special about them Need a special polymerase though Taq DNA polymerase is used T (thermus) aq (aquaticus) hot water it is the type of bacteria that the polymerase comes from, it was isolated from a geyser in Yellowstone, it survives in water near boiling because all of its proteins are used to this environment and unusually resistant to denaturation by heat, it is an example of a thermophile The reason you want Taq polymerase because the technique uses a very high temperature over and over again and if you used a regular polymerase it would denature every time you raise the temperature and youd have to continuously add more polymerase after every heating Mix template, primers (ssDNA), dNTPs, and Taq DNA polymerase in aqueous solution and put it in a thermal cycler (like a hot plate that is programmed to hold the solution at certain temperatures for certain lengths of tim) Thermal cycler controls the temperature of the solution Temperature of solution controls the events of this modified replication There are three steps Denaturation at 93 degrees C o Gets the DNA to be single stranded, instead of needing to use helicase Annealing at 50 degrees C (approximately) o This colder temperature allows hydrogen bonding between the primers and their complimentary sequences, allows for annealing to the template o This temperature is variable because the different times you do PCR the primers are different sequences and you have to tailor the temperature to the primers you are using o Taq polymerase seems to have what it needs, template, 3 end, dNTPs, but 50 degrees is not a good temperature for Taq polymerase it doesnt operate optimally here o DNA pol deltas ideal temperature is our body temperature 37 degrees o DNA pol IIIs ideal temperature is also 37 degrees, same with DNA pol I...
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