PCR reac on mix, 8 tubes, 50 μL each: Mix contains Taq DNA polymerase, MgCl2, and dNTP’s
DNA/ primer mix, 8 tubes, 50 μL each: Each mix contains a different template DNA. All tubes
Same forward primer and reverse primer
A strip of empty PCR tubes
Disposable pipets
Cup for discarded tips
Micropipette
OpenPCR machine (shared by two groups)
PCR Reaction Sample List
Tube Label
PCR Reaction Sample
Patient ID
G9 +
Positive control
none
G9 -
Negative control
none
G9 1-1
Patient 1, replicate 1
17649
G9 1-2
Patient 1, replicate 2
17649
G9 1-3
Patient 1, replicate 3
17649
G9 2-1
Patient 2, replicate 1
28137
G9 2-2
Patient 2, replicate 2
28137
G9 2-3
Patient 2, replicate 3
28137
DNA Sample Set-up Procedure
First, place the extracted piece of DNA into the special PCR tube
Add Primer 1 to the PCR tube (first binding site)
Add Primer 2 to the PCR tube (second binding site)
Add Nucleotides to the PCR tube (A's, C's, T's, and G's)
Add the DNA Polymerase into the PCR tube (matches nucleotides)
Place into DNA Thermal Cycler
Begin Thermal Cycler
Allow the cycles (about 3) to carry on from 95 degrees Celcius to 50 degrees Celcius and then 72 degrees Celcius
OpenPCR program
Description of the Thermal Cycle
Research and Development
PCR - The Underlying Technology
Components Of PCR
The main components of PCR include Template DNA, Primers, Taq Polymerase, and Deoxyribonucleotides (dNTP’s). The Template strand is the basis for this experiment. The Deoxyribonucleotides are supplied to the Taq polymerase in order to replicate the template DNA. Once this is achieved, Taq polymerase makes new strands of DNA. These new strands of DNA contain different, specific base pairs that the primers will locate and attach to. Essentially, Primers are used to determine the DNA fragment to be amplified during PCR. This makes differentiating the replicated base pairs that are attached to PCR easy as they continue to be copied.
Thermal Cycling
Base-pairing occurs between the annealing and extension steps of the thermal cycle. During the annealing stage, the strand is cooled to 57 degrees Celsius so the primers can bind to their complementary sequences on the single-stranded template DNA. Then in the extension stage, the temperature is raised to 72 degrees Celsius and Taq polymerase extends the primers, synthesizing new strands of DNA, which means base pairs are matched with their complementary base to form new strands. A nucleotide consists of a base, a five-carbon sugar, and a phosphate group and is the basic unit that makes up DNA. A polymorphism is any difference in a nucleotide sequence between individuals, so basically a DNA sequence variation.
Annealing
Once the base pairs begin to anneal (come together or pair up), they connect with their specific complementary base. The pairs are driven by Hydrogen Bonding which explains the attraction that they have. Adenine pairs with Thymine while Guanine pairs with Cytosine.
Experiment
The positive control is DNA from a person who tested positive for the disease SNP. The negative control is DNA from a person who does not carry the disease SNP. Patients 1 and 2 have never been tested before.
SNP Information & Primer Design
Background: About the Disease SNP
The disease SNP has a variation in Homo-Sapiens with an uncertain clinical significance. This is caused by many different reasons. The variant is located on chromosome 12 which links this to Parkinson's Disease. Specifically, it is 40315266. The disease-associated allele contains the codon GAG. Because this is a variant of the nucleotide sequence, it is called a polymorphism. The nucleotide is composed of the bases Adenine, Thymine, Guanine, and Cytosine. Adenine pairs with Thymine and Guanine pairs with Cytosine. If there is a frame-shift, the base pairs move over and change all the other pairs because of their specific pairs.
Primer Design and Testing
After testing the Primer, the result of "No Match" was displayed which correlated to 220 bp. This is because the data base only tests for human gene sequences that do not contain the SNP variations. Thus, the experiment went as expected and was successful.
BME 100 Fall 2018
