BME100 f2014:Group20 L4
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LAB 4 WRITE-UP
* Taq DNA polymerase * MgCl2 *dNTP
PCR Reaction Sample List
Research and Development
PCR - The Underlying Technology
Function of Components
- Primers: Primers are short single strands of DNA that are manufactured in a laboratory. They are designed to have complementary nucleotides to the the sequence of DNA that will be replicated during PCR. One primer goes on the top of the DNA sequence on one strand and the other primer goes on the end of the DNA sequence of the opposite strand. Primers serve as a starting place for DNA polymerase as it requires a double strand to begin adding nucleotides.
- DNA Polymerase: DNA Polymerase is a protein complex that is used to copy DNA for a cell before mitotic devision. DNA polymerase travels along the DNA strand of replication until it comes in contact with a primer. Once it does, it begins assembling the new DNA strand by adding complementary nucleotides to the original strand.
- Nucleotides: Nucleotides used in PCR are what form the "rungs" of the double helix shape of DNA. There are four nucleotides that DNA polymerase used while assembling DNA in PCR; they are adenine, thymine, guanine, and cytosine. They are located around the newly forming DNA strand and DNA polymerase pulls them in to match to the nucleotides on the original strand.
1. Sample DNA is extracted from a cell
2. Extracted DNA is put into PCR tube via micropipette
3. Primer 1 is added to the PCR tube and binds to site 1
4. Primer 2 is added to the PCR tube and binds to site 2
5. The nucleotides are added to the PCR tube
6. DNA polymerase is added to the PCR tube
7. The PCR tube is put into a DNA Thermal Cycler
8. The Thermal Cycler sets the temperature to 50 degrees Celsius and the DNA strands try to pair up but the primers prevent them from doing so and bind to the ends of the replication sequences
9. The temperature is raised to 72 degrees celsius and DNA polymerase is activated. DNA Polymerase locates a primer and assembles DNA until it reaches the end of the strand and falls off
10. Steps 8 and 9 are repeated 29 more times and a billion DNA fragments are created (most are the pure sequence, but 60 contain longer sequences)
Each nucleotide is composed a phosphate, deoxyribose, and 1 of 4 nitrogenous bases. The 4 bases are adenine, thymine, guanine and cytosine. Adenine and Guanine are purines (double ring) and Thymine and Cytosine are pyrimidines (single ring). Adenine binds with thymine and guanine binds with cytosine. They bind to each other through hydrogen bonds due to intermolecular forces. Adenine and thymine have a double bond and cytosine and guanine have a triple bond. The manner in which these nitrogenous bases bind to each other is what causes DNA to have the double helix shape.
Image obtained from http://academic.brooklyn.cuny.edu/biology/bio4fv/page/genetic-engin/pcr.html