BME100 f2015:Group17 8amL4
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Lab Write-Up 1 | Lab Write-Up 2 | Lab Write-Up 3
Lab Write-Up 4 | Lab Write-Up 5 | Lab Write-Up 6
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LAB 4 WRITE-UP
PCR Reaction Sample List
have the same forward primer and reverse primer
samples will be cross-contaminated
extend at 72°C for 30 seconds
Research and Development
PCR - The Underlying Technology
The Process of Thermal Cycling: First, the DNA is placed in a thermocycler. The thermocycler raises the temperature of the DNA to 95 degrees Celsius for three minutes. After the DNA double helix has been kept at this temperature for this time, a thirty second period then begins in which two single-stranded DNA molecules are created. These two single-strands are a result from the separation of the original double helix. After this process, the DNA is brought down to a temperature of 57 degrees Celsius for thirty seconds. During this time period, primers attach to DNA sites so the two single strands do not bond together again. Once the primers are attached, the thermocycler changes temperature to 72 degrees Celsius for 30 seconds. This temperature change activates DNA polymerase. Finally, after three more minutes of sitting in 72 degrees Celsius, nucleotides are added to the single-stranded DNA by the primers. The primer follows the entire strand to add nucleotides. The temperature then changes to four degrees Celsius to halt the process.
Adenine (A) pairs with Thymine (T). Cytosine (C) pairs with Guanine (G). Guanine (G) pairs with Cytosine (C). Thymine (T) pairs with Adenine (A).
SNP Information & Primer Design
Background: About the Disease SNP The two steps of thermal cycling during which base pairing occurs are “Annealing” and the “Extension” stages. During the Annealing stage the temperature is lowered to 57 degrees celsius. At this temperature the DNA strands are looking to recombine however the massive amount of primer 1 (a specific sections of the DNA) present causes the DNA strands to bind to the primers at the top strand at one end of the segment of interest. At the Extension stage, the second primer also binds to the bottom strand at the other end of the DNA strand.
This variation is found in Homo sapiens. The variation is located on the chromosome, 16:89919736. The Clinical significance of this SNP is pathogenic. SNP is associated with the gene, MC1R. MC1R stands for Melanocortin 1 receptor. The MC1R gene provides instructions for making a protein. An allele is an alternate form of a gene caused by a mutation. They are found at the same place on a chromosome.
Primer Design and Testing
The objective of this process was to understand how base pairs are altered within the human genome. This includes mutations from unwanted base pairs. The process illustrated what steps are taken when base pairs are changed. This included the change from CGC to CGG.