electrophoresis_module_sept28_2003

electrophoresis_module_sept28_2003 - 1 What if...there is a...

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Unformatted text preview: 1 What if...there is a mutation or polymorphism? What if we had a polymorphism or mutation? How would we detect it? Procedure Step 1 : Isolate the gene by using primers. Remember, the complementary DNA strand is defined with a different primer on the 5 end. Below is a picture of both a typical gene and a variant (i.e., polymorphism). T C G A G C G A T G A T T A C G G A A C C G A C C A A G T C A T C G T C G A G C G A T G A T T A C G G A T T T G A C C A A G T C A T C G Primer Primer Typical Variant 1 Mutation 500 bp 500 bp 5 3 5 3 2 Step 2: Develop an enzyme, a restriction enzyme, that will cut the gene at mutation TTT. T C G A G C G A T G A T T A C G G A A C C G A C C A A G T C A T C G T C G A G C G A T G A T T A C G G A T T T G A C C A A G T C A T C G Primer Primer Typical Variant 1 300 bp 200 bp The restriction enzyme will cut or cleave the gene at mutation TTT 500 bp 5 3 5 3 3 Step 3: Introduce the restriction enzyme to the typical and the variant genes. Result 1: The 'typical' gene will remain a single 500 bp fragment. Result 2: The 'variant' gene will be cleaved into 2 fragments at the mutation: one 200 bp fragment one 300 bp fragment Result 3: The TTT mutation will be in the 200 bp fragment. T C G A G C G A T G A T T A C G G A A C C G A C C A A G T C A T C G T C G A G C G A T G A T T A C G G A Primer Primer Typical Variant 1 300 bp 200 bp The restriction enzyme will cut or cleave the gene at mutation TTT 500 bp T T T G A C C A A G T C A T C G 5 3 5 3 4 Seeing is believing How do we demonstrate that the procedure has indeed worked? Once we have cut the original variant 1 gene (500 bp) into two fragments of 200 and 300 bp, we can use gel electrophoresis to help us visualize the two fragments. The fragments will be pushed to the end of the gel lane by the electrical charge in order the two fragments....
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electrophoresis_module_sept28_2003 - 1 What if...there is a...

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