BME100 f2015:Group1 1030amL4
|BME 100 Fall 2015|| Home |
Lab Write-Up 1 | Lab Write-Up 2 | Lab Write-Up 3
Lab Write-Up 4 | Lab Write-Up 5 | Lab Write-Up 6
Course Logistics For Instructors
Wiki Editing Help
LAB 4 WRITE-UP
PCR Reaction Sample List
Research and Development
PCR - The Underlying Technology
Thermal Cycling: The PCR process from start to finish takes 5 steps to complete. Step one: the separation step, is where the DNA strand is heated to 95°C, allowing for the DNA to Denature and separate into single stranded molecules.The second step is the start of the bonding process, where the Primers bond to the specific DNA strand segments that we want to copy. The temperature is lowered to 57°C for this process, in order to allow the primer to bond. The third step is: the temperature is then raised to 72°C to active the DNA polymerase and allow it to bind to the DNA strand and the primer. The fourth step: At 72°C again,the DNA strand is transcribed, creating our copied segment. The fifth and final step is when new primers are attached to both the copies and the template strand, allowing for the PCR process to repeat and more copies to be created.
Nucleotide Base-Pairing: The four nucleotides are Adenine, Thymine, Cytosine, and Guanine, and work to build base pairs in the DNA strand. Adenine binds to Thymine, and Cytosine binds to Guanine.The base pairs are formed in the 3rd and 4th steps of the PCR process, because in steps 3 and 4, the primers are places on the template strand, and the Polymerase binds to both the primer and the template strand.
File:PCR Process Pictures Group1 Lab4 1030am.docx Images located from:
DNA Learning Center (2010, March 22). Polymerase Chain Reaction (PCR) [Video file]. Retrieved from https://www.youtube.com/watch?v=2KoLnIwoZKU
SNP Information & Primer Design
Background: About the Disease SNP The single nucleotide polymorphism (SNP) disease is rooted in the nucleotides of the DNA itself. SNP is when a single nucleotide (a nucleotide is the monomer of which DNA is composed) is replaced by another nucleotide. For example, SNP is when a cytosine base is replaced by a guanine base. SNP is the most common genetic variation, however most SNP mutations have no affect on the gene expression and the health of the organism. But that does not mean SNP does not lead to diseases. In fact SNP can lead to a whole range of diseases , ranging from autoimmune diseases such as Celiac Disease, to simpler disorders such as glaucoma. The primer given in the lab is a primer for Homo sapiens. The variation occurs on the 16th chromosome, and is associated with the MC1R gene. The Clinical significance of the SNP is that it is pathogenic. The disease associated with this mutation is Parkinson Disease and susceptibility to Clear Renal Cell Carcinoma.
Primer Design and Testing The disease related SNP is located on the top strand of the DNA sequence, which means it is going from 5' to 3' with a 20 base long sequence. The non-disease forward primer was 5'-CGGGCGCGGCGAGCCGTTGC. The second primer was the non-disease reverse primer, which is located exactly 200 bases to the right of where the diseased SNP was located. This numerical position is 89919936. The non-disease reverse primer was 5'-CTTGTGGAGCCGGGCGATGC. Next, a forward and reverse primer for the disease was needed. The Process was similar except the final base on the non-disease forward primer was replaced with the disease related base found in the SNP. The disease forward primer happened to be 5'-CGGGCGCGGCGAGCCGTTGT. Afterwards was the disease reverse primer which ended up being 5'-CTTGTGGAGCCGGGCGATGC. When the primers were checked using the UCSC In-Silico PCR website our data came back with error. The sequence had a 201 bp which is not valid compared to the 220 bp that it should be. This error could come from entering a wrong base in the sequence or possibly not having the correct primer in the system, which in return would cause some of the data to be off. Lastly, the image of the diseased primers came back as not matching. This could be because of some fragments within the sequence.
This image of the PCR program shows that there was not a match.