Lecture26

Lecture26 - BIS101/Engebrecht Lecture26 6/1/10 PCR...

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BIS101/Engebrecht Lecture26 6/1/10 PCR (cont).What you need: 1) target DNA – you only need a small amount of DNA, for instance, a single cell 2) set of primers – these are short stretches of single stranded nucleotides that are synthesized by organic chemistry that are homologous to your gene of interest. You design them to base pair with the sequence you are interested in (which means you need sequence information). 3) DNA polymerase called Taq polymerase because it is from a thermophilic (heat loving) organism – Thermophilus aquaticus . This organisms lives at very high temperatures so that the polymerase survives multiple rounds of heating. 4) dNTPs, for the synthesis of the new DNA molecules Briefly, you place the above components into a test tube in a “PCR machine”. The machine cycles rapidly between different temperatures as follows: ~95 0 C, to denature the target DNA ~60 0 C, to allow the primers to anneal to the ssDNA that they are homologous to. This now provides the substrate for Taq to synthesis new DNA. ~72 0 C, to allow the polymerase to extend or polymerize the new strand of DNA complementary to the target DNA This method is exquisitely sensitive. Starting with a single target molecule, after 10 cycles you have 1014 copies, after 20 cycles you have greater than a million, after 30 cycles you have greater than a billion copies of your DNA (2 n , where n = the number of cycles). CQ1: Only the first pair of primers (answer = A ) can bind to the target DNA and direct amplification of the desired DNA molecule. A former student has also set up a web page that describes PCR (http://ucd-pcr.com/default.aspx) that you may find useful. Reverse Genetics – making mutants Since we have the entire human genome sequenced (as well as other organisms), reverse genetics is currently the prevalent approach to analyzing gene function. To undertake such a task, we need ways to generate mutants for each gene so that we can examine the resulting phenotype of an organism that is mutant for that gene. The ability to generate mutants relies on: 1) cloned piece of DNA 2) way to introduce DNA into cells (We have already seen that you can introduce DNA into a cell by transformation and via viruses. You can also directly inject DNA or shoot DNA into a cell by using a gun.) 3) homologous recombination between cloned piece of DNA and chromosomal DNA The yeast, Saccharomyces cerevisiae , has been a model system for establishing techniques used for reverse genetics and genomics. In yeast, it is quite easy to generate “knockout” strains, that is, yeast strains that are deleted for a gene. In class, I used the URA3 gene as an example of a selectable marker. In vitro, by the techniques of cloning, you can generate a piece of DNA that has your gene deleted and replaced with URA3 . This piece of DNA can be introduced into yeast and cells that have undergone homologous recombination can be selected for on medium lacking uracil.
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Lecture26 - BIS101/Engebrecht Lecture26 6/1/10 PCR...

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