BME100 s2017:Group4 W8AM L4
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LAB 4 WRITE-UP
have the same forward primer and reverse primer
do, the samples will become cross-contaminated
PCR Reaction Sample List
HEATED LID: 100°C
INITIAL STEP: 95°C
NUMBER OF CYCLES: 25 (Denature at 95°C for 30 seconds, Anneal at 57°C for 30 seconds and Extend for at 72°C for 30 seconds)
FINAL STEP: 72°C for 2 minutes
FINAL HOLD: 4°C
Research and Development
PCR - The Underlying Technology
The Template DNA is used as a guide to make the replicate DNA. Primers attach to sites on the DNA strands that are at either end of the segment that is being copied. The Taq DNA Polymerase reads the DNA code and attaches matching nucleotides to create DNA copies. The deoxyribonucleotides (dNTP’s) are genetic building blocks that are used to create the replicate DNA strands.
During the initial step at 95°C for 3 minutes, the DNA double helix separates, creating two single-stranded DNA molecules. During the denature step at 95°C for 30 seconds, the DNA double helix separates, creating two single-stranded DNA molecules. During annealing at 57°C for 30 seconds, the primer sequences bind to specific areas on the single-stranded DNA. During it's extension at 72°C for 30 seconds, the DNA polymerase finds the primer and attaches to the DNA strand and begins to add complementary nucleotides onto the strand. During the final step at 72°C for 3 minutes, the DNA polymerase finishes adding to the DNA strand and falls off. During the final hold at 4°C, the DNA stand cools down to ensure that everything has recombined fully.
there are four nucleotides that are the basis of the DNA molecule. The four nucleotides are adenine, thymine, cytosine, and guanine. Due to the number of available spaces for hydrogen bonding, adenine binds with thymine and cytosine binds with guanine.
Base-pairing occurs during annealing and extension. In annealing, the nucleotides of the primers bind with their complementary base pairs on the DNA strands. In extension, DNA polymerase attaches complementary base pairs to the base nucleotides on the DNA strand.
In order for the primers to be able to bind with the cancer DNA template, the template DNA must first be denatured which produces two complementary single-stranded DNA molecules.
Once the DNA is denatured and the temperature is lowered to the optimum temperature, the primers pair up with specific sites on the template DNA where the nucleotides of the DNA are perfectly complementary to the nucleotides of the primer.
The Taq polymerase then binds to the primers that have attached to the sites on the template DNA in preparation for amplifying the DNA.
The Taq polymerase begins moving along the template DNA, attaching complementary nucleotides to the DNA as it progresses. Once the Taq polymerases have finished the template DNA strand, they detach and the DNA has been amplified.
(Pictures taken from http://learn.genetics.utah.edu/content/labs/pcr/)
SNP Information & Primer Design
Background: About the Disease SNP
SNP stands for "single nucleotide polymorphism." A nucleotide is an organic molecules that serve as the monomer units for forming the nucleic acid polymers DNA and a polymorphism is a common variation in the sequence of DNA among individuals. Genetic variations occurring in more than 1% of the population would be considered useful polymorphisms for genetic linkage analysis. When searching our specific SNP, it was found that the condition linked to this one was cystic fibrosis. This is found in homo sapiens and is located on the chromosome 7:117587799. Under the GeneView of this disease, we found out that CTFR stands for cystic fibrosis transmembrane conductance regulator. The non-disease allele associated with this is AGT, and the disease-associated allele is CGT.
Primer Design and Testing
The numerical position of the SNP is 117587799. The non-disease forward primer was found to be 5’- A G A A G G T G G A A T C A C A C T G A, while the non-disease reverse primer was found to be 5’- C A T T A T T T A T A G T T C T T A A A. The disease forward primer was found to be 5’- A G A A G G T G G A A T C A C A C C G T and the disease reverse primer was 5’- C A T T A T T T A T A G T T C T T A A T. When ran through the In-Silico PCR website, the non-disease primer resulted in 220bp sequence from the chromosome indicating the primer worked because it is a known sequence. The disease- specific primer designed resulted in no matches, because of a mutation in the DNA sequence, which matches what was expected of the diseased primer.
"PCR Virtual Lab" http://learn.genetics.utah.edu/content/labs/pcr/