Bio 97 Lecture 3.docx

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Biological Sciences
Rahul Warrior

PCR • Requires a DNA template • Requires a sequence-specific primer—you have to know at least part of the DNA sequence • Requires nucleotide triphosphates • Requires a special polymerase • Is semiconservative DNA separates into two strands of DNA by heating to 95C G-C has 3 H-bonds and A-T have 2-H bonds so diff. organisms have different heating points After separating, you cool the DNA and the short primers that are between 16-25 bases long and they base pair to the two single strands Each of the strands will base pair with a primer Raise the temperature slightly so the DNA polymerase will start off at the primer and copy the two strands. One double strand à two double strand Denature à anneal à extend Need DNA, template, and DNA poly Semi conservative • 95°C→denatures DNA by breaking the hydrogen bonds between strands • 50-65°C→temperature is reduced to allow primer to bind (anneal) to DNA • 72°C→temperature is raised to the optimum for the thermostable polymerase (Taq) to extend the DNA strand • Similarities to replication and PCR • Requires a template • Requires primers • Requires nucleotide triphosphates • Requires a polymerase Dideoxyribose cannot extend the chain. You need a hydroxide group to extend the chain Phosphidiester bond with nucleotide and the hydroxyl group (OH group) By incorporating a dideoxyrbiose into the chain, you stop the replication, bc the next nucleotide cannot form a bond with a H. It needs an O. With a molecule with all dideoxyribose, the replication would terminate But you don’t need all of them to be dideoxy but just a few of them for the strands to be different lengths There are four different colors for each dideoxytriphosphates 1 out 20 is a dideoxy Occasionally instead of incorporating a deoxy, you incorporate a dideoxy so the replication can stop. If you color of the molecule, the last chain will be a color bc you end with a dideoxy If you see a molecule with a black, then you will know that it ends with an G (example) The color correlates with the l
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