BME100 f2014:Group23 L4
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LAB 4 WRITE-UP
The OpenPCR program is contained in the computer in the thermo cycler. The information above contains all the data that is inputed into the system. The process begins when the temperature is raised to all the DNA to denature and to split apart into to separate strands. This is the Initial Step, heating the solution to 95 degrees celsius. Then, the DNA is cooled, and the primers added to the solution attach to their binding sites on the DNA, this it the "Anneal" portion described above, at 57 degrees celsius. After this, taq DNA polymerase begins to add nucleotides to the DNA, replicating it. This process is in the "Extend" portion in the data above, at 72 degrees celsius. This is then repeated in a certain amount of cycles to exponentially increase the amount of replicated DNA of that specific portion selected by the primers. This exponential increase is called amplification in the DNA Polymerase process.
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PCR - The Underlying Technology
What Happens During Thermal Cycling During the first step, the components are heated to 95 degrees Celsius for three minutes. Then the components are heated at 95 degrees Celsius for another thirty seconds. During this time, the two strands of DNA separate, or denature. Then the components cool to 57 degrees Celsius, when the two primers bind, or anneal. Each primer binds to a different strand of DNA, at opposite sides of the strands. Then the components are heated to 72 degrees Celsius for thirty seconds, when the DNA polymerase binds to the end of the primers, and begins extending along the DNA strand. This process is completed as the temperature is kept stable at 72 degrees Celsius for three minutes, while the polymerase attaches the remaining nucleotides to complete the two double stranded DNA molecules. During the final hold, the components are cooled to 4 degrees Celsius. During this time, the replication is complete and two double stranded DNA molecules have formed.
Which Bases Anneal to Which Bases Only certain bases can attach to eachother. There are four bases, including Adenine, Thymine, Cytosine, and Guanine. Adenine and Thymine can only bond with one another. Likewise, Cytosine and Guanine can only bond with one another.
When Base Pairing Occurs
Base pairing occurs during two steps of the PCR process. It occurs during both the forth and fifth steps, when the components are heated to 72 degrees Celsius for the first thirty seconds, then for three minutes. This is because at this point, both the primer and the DNA polymerase have attached to the DNA strands, allowing for nucleotides to be attached to the DNA strands by the polymerase. During the forth stage, the polymerase attaches to the DNA strand and begins base pairing, but an additional three minutes at the same temperature of 72 degrees Celsius during stage five are necessary to finish up the process.