Each component of the PCR must be in coordination with one another in order to accurately multiply the DNA sequence that is needed. The template DNA is the whole of the DNA from which the single DNA sequence that is needed will be copied from. The primers attach to each end of the nucleotide and serve as promoters of the DNA replication process. The Taq polymerase targets the primers attached to the DNA strand and adds complimentary nucleotides onto the strand. The deoxyribonucleotides attach to form the DNA.
Once all of the components are added to the PCR tube, the tube is inserted into the DNA thermal cycler. At 95 degrees Celsius for two minutes the DNA splits apart into two single stranded DNA pieces. The denature step serves to extend the initial step and further the PCR reaction allowing the DNA strands to be completely seperated. When the reaction goes through the anneal phase at 57 degrees Celsius for 30 seconds, the single stranded DNA molecules naturally attempt to pair up, but the primers crowd their way in and lock onto their target before strands can rejoin. When the thermal cycler is heated up to 72 degrees Celsius for 30 seconds, it begins the extend phase within the PCR reaction. This triggers the DNA polymerase to locate a primer attached to a single DNA strand, it begins to add complimentary nucleotides onto the strand. It continues until it gets to the end of the strand and falls off. For the final step at 72 degrees Celsius for 2 minutes, the cycle repeats over and over again and the desired fragments begin to appear. fragments multiply until there is an almost pure solution of the target DNA. At 4 degrees Celsius the DNA is held in place.
DNA is made up of four types of molecules called nucleotides designated as A,T,C, and G. These Base-pairs are driven by hydrogen bonding, allowing the base pairs to stick together. The base pairs anneal to each other, Adenine (A) anneals to Thymine (T) while Guanine (G) anneals to Cytosine (C). Base-Pairing occurs during the anneal at 57 degrees for 30 seconds and the extend at 72 degrees for 30 seconds.
SNP Information & Primer Design
1. Background: About the Disease SNP
What is a nucleotide?
-A nucleotide is a compound consisting of a nucleoside linked phosphate group.
What is a polymorphism
-A polymorphism is a common variation in the sequence of DNA among individuals.
What species is this variation found in?
-The species this variation is found in is Homo Sapiens.
What chromosome is the variation located on?
-The variation is located on chromosome 19: 44907853.
What is listed as the clinical significance of this SNP?
-The clinical significance of this SNP is pathogenic.
What condition is linked to this SNP?
-This SNP is linked to Alzheimer's and Subarachnoid Hemorrhage.
What does APOE stand for?
-APOE stands for apoliprotein E.
What is the function of APOE?
-APOE provides instructions for making the apolipoprotein E which combines with lipids in the body to create lipoproteins which are responsible for bundling cholesterol as well as carrying it through the blood stream. Maintaining the right levels of cholesterol is essential for the prevention of cardiovascular diseases such as heart attack and stroke. The major alleles are E2, E3, and E4.
What is an allele?
-An allele is one of two or more alternative forms of a gene that arise by mutation and are found at the same place on a chromosome.
2. Primer Design and Testing The disease associated allele contains what codon?
-The disease associated allele contains the CCG codon.
The numerical position of the SNP is: 44907853.
The non-disease forward primer is 5'AGCGGCCAGCGCTGGGAACT.
The numerical position exactly 200 bases to the right of the disease SNP is: 44908053.
The non-disease reverse primer is 5'AGGCCCCCCAAGACTTAGC.
The disease forward primer is 5'AGCGGCCAGCGCTGGGAACC.
The disease reverse primer is 5'CAGGCCCCCCAAGACTTAGC.
BME 100 Fall 2017
