PCR reaction mix, 8 tubes, 50 microliters each: Mix contains Taq DNA polymerase, MgCl2, and dNTP's
DNA/ primer mix, 8 tubes, 50 microliters each: Each mix contains a different template DNA. All tubes have the same forward primer and reverse primer
A strip of empty PCR tubes
Disposable pipette tips: only use each only once. Never reuse disposable pipette tips. If you do, the samples will become cross-contaminated
Cup for discarded tips
Micropipettor
OpenPCR machine: shared by two groups
PCR Reaction Sample List
Tube Label
PCR Reaction Sample
Patient ID
G3 +
Positive control
none
G3 -
Negative control
none
G3 1-1
Patient 1, replicate 1
83982
G3 1-2
Patient 1, replicate 2
83982
G3 1-3
Patient 1, replicate 3
83982
G3 2-1
Patient 2, replicate 1
29244
G3 2-2
Patient 2, replicate 2
29244
G3 2-3
Patient 2, replicate 3
29244
DNA Sample Set-up Procedure
Acquire proper lab attire
Label each of the 8 clean PCR tubes using the labels in the table
Acquire a clean tip for the micropipettor
Using the micropipettor, transfer 50 microliters of PCR mix into each of the 8 labeled PCR tubes
Discard the used tip into the cup and obtain another clean tip for the micropipettor after each use
Using the micropipettor, transfer 50 microliters of the "G3+" DNA/ primer mix into the PCR tube labeled "G3+"
Repeat step 6 for each of the remaining 7 PCR tubes, discarding the used tip and obtaining a clean tip after each use
OpenPCR program
Heated Lid: 100 degrees Celsius
Initial Step: 95 degrees Celsius- DNA begins to unravel
Number of Cycles: 25
Denature at 95 degrees Celsius for 30 seconds- DNA separates into two single-stranded DNA molecules
Anneal at 57 degrees Celsius for 30 seconds- Pairs up with primers
Extend at 72 degrees Celsius for 30 seconds- Activates DNA Polymerase
Final Step: 72 degrees Celsius for 2 minutes- DNA Polymerase locates primer and adds complementary nucleotides
Final Hold: 4 degrees Celsius- Reaction solidifies
Research and Development
PCR - The Underlying Technology
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.
This picture shows that both the forward and reverse primers work and that they are correct, which means that the diseased primers are also correct.
BME 100 Fall 2017
