BME100 f2014:Group14 L4
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LAB 4 WRITE-UP
Denature at 95°C for 30 seconds, Anneal at 57°C for 30 seconds, and *Extend at 72°C for 30 seconds
Research and Development
PCR - The Underlying Technology
The Function of Each Component of a PCR Reaction:
The purpose of the Template DNA is to be the original DNA for replication. Without original DNA, there would be no way to select specific sections for replication, since all DNA is replicated from existing DNA. Small samples of Template DNA can be extracted from blood, skin or hair follicles. Primers are small synthesized sections of DNA that are designed to attach to a certain sequence of base pairs on the Template DNA. They single out the section of the Template DNA for replication and the enzyme Taq Polymerase uses it as a tag to start replication. Without the primers, Taq polymerase has nowhere to attach so no DNA replication would occur. Primers bond to specific sequences of he Template DNA based off of base pairs. Since A binds with T and C binds with G, the specific primer sequence will match up with the specific DNA pair, singling out the targeted gene. The purpose of the enzyme Taq polymerase is to replicate the specific part of the Template DNA. It attaches to the primers and begins to synthesize a replicated strand of DNA once the PCR is heated to 72 °C. Taq polymerase is found in bacteria that live in extreme heat conditions making it resistant to denaturing by the PCR heat cycles. It is not found in humans. When Taq polymerase is activated, it does not stop synthesizing the new strand until it reaches the end of the Template strand. Deoxyribonucleotides are the A T C and G nucleotides that make up the DNA. Since A binds with T and C binds with G, the Taq Polymerase uses the excess deoxyribonucleotides to match up to the Template DNA. Deoxyribonucleotides include all the sections of the DNA including the nitrogen base, the deoxyribose sugar and the nucleotide. These three components comprise the whole strand of DNA and are necessary to make the replicated strands.
Component Behavior During Steps of Thermal Cycling:
During the initial step where the reaction is heated to 95°C for 3 minutes to heat the PCR machine. Then, the Template DNA is initially denatured for 30 seconds meaning the two strands that make up the DNA separate. The primers, Taq Polymerase and the Deoxyribonucleotides are inactive at this stage. The next step is to Anneal at 57 °C for 30 seconds. This cooling step allows the primers to attach to the Template DNA and because there are so many primers in the solution, the primers attach more quickly to the Template than the two strands of DNA could recombine. The Taq Polymerase and Deoxyribonucleotides are still inactive at this stage. The next step is to Extend at 72 °C for 30 seconds which allows the Taq Polymerase to start replicating using the Primers as a starting point. The Taq Polymerase builds the replicated strand of DNA using the Deoxyribonucleotides in the solution. These steps are repeated 35 times. The Final Step is 72 °C for 3 minutes ensures that every Template strand has been replicated and the Final Hold preserves the structure of the DNA.
Adenine (A) binds with Thymine (T) and Cytosine (C) binds with Guanine (G.) During the Anneal and Extend steps of PCR, base pairing occurs. In the Annealing step, the primers pair up with the Template strands and during the Extending step, Taq Polymerase pairs the Deoxyribonucleotides with the Template strands to synthesize the new strand.