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 have the same forward primer and reverse primer
A strip of empty PCR tubes
Disposable pipette tips
Cup for discarded tips
Micropipettor
Open PCR machine
PCR Reaction Sample List
Tube Label
PCR Reaction Sample
Patient ID
G15 +
Positive control
none
G15 -
Negative control
none
G15 1-1
Patient 1, replicate 1
40686
G15 1-2
Patient 1, replicate 2
40686
G15 1-3
Patient 1, replicate 3
40686
G15 2-1
Patient 2, replicate 1
68520
G15 2-2
Patient 2, replicate 2
68520
G15 2-3
Patient 2, replicate 3
68520
DNA Sample Set-up Procedure
Collect all materials listed above.
Cut the strips of PCR tubes in half so you have 2 equal sections of 4
Using a fine point black maker label the tubes on the side as noted above,(G15+. G15-, G15 1-1, Ect.)
Place the tubes in the appropriate PCR tube rack
Find the tube labeled as Positive control, G15+, and transfer 50 µl of PCR reaction mix into the tube, make sure to use proper pipetting technique, discard tip after each use to make sure we do no cross contaminate the DNA samples.
Transfer 50 µl of Positive control DNA/ Primer mix with a clean pipette tip into the same tube, bringing the total volume to 100µl for the positive control, G15+, tube.
this will result in all labeled tubes containing 100 µl
Close the lids tightly on all of the PCR tubes
take the tubes to the designated PCR machine, DO NO START until all slots are filled with the other groups.
OpenPCR program
Research and Development
PCR - The Underlying Technology
Component Functions
The PCR reaction consists of four different types of components including Template DNA, primers, Taq Polymerase, and Deoxyribonucleotides (dNTP's). The function of each varies greatly to be able to successfully copy a segment of DNA billions of times. The DNA template is the sample DNA used to give the needed target sequence. The primers (single nucleic acid sequence) serve as start points for DNA synthesis to occur. Taq polymerase is an enzyme that synthesizes the DNA into new strands. It is what binds the complementary nucleotides to their partner on the target sequence. Deoxyribonucleotides are the components needed to build new DNA strands. These include adenine, thymine, guanine, and cytosine.
Components during Thermal Cycling
Thermal cycling happens in a set of six general steps. The Initial Step consists of the DNA Template being heated to 95°C for 2 minutes. This is the time when the DNA is heated to begin the process of PCR. Following this is the Denaturization at 95°C, still, for 30 seconds. What happens is the DNA's double strand gets separated into single strands. The next step is Annealing at 57°C for 30 seconds. This annealing allows for the primers to attach to the single-stranded DNA templates. Two primers are added, one for each single-strand, to its designated, complimentary match on the target DNA sequence. They do this in order to be able to be copied later. Extension occurs next at 72°C for 2 minutes. At this point, the enzyme, Taq Polymerase, makes an appearance. It binds to the prime sequence and adds free dNTP's to their correct complement in the 5'-3' direction to extend the strand. Second, to last, the Final elongation happens at 72°C, again, for 2 minutes. This ensures that the previous events happened and make sure any single-stranded DNA is completely elongated. In the final step, the final hold, the reaction is cooled to 4°C so that the cycle stops and no more unnecessary replications are made.
Nucleotides and Base Pairing
Nucleotides is the name for the four types of molecules that make up DNA. They are known as Adenine (A), Thymine (T), Guanine (G), and Cytosine (C). When base-paired, or stuck together by hydrogen bonding, the A and T stick together while the G and C stick together. Base-pairing occurs during the annealing step as this is where the primers begin to bind to the complimentary matches on the single-strand target DNA sequence. Base-pairing also happens in the extension step when the enzyme binds to the prime sequence and adds the appropriate complements to the second strand.
Original PCR Illustration
SNP Information & Primer Design
Background: About the Disease SNP
A single nucleotide polymorphism, or SNP, is a change in a single nucleotide that can act as a red flag or indicator for a disease. The disease linked to our SNP is Parkinson's disease and the SNP is listed as uncertain clinical significance. The disease is found in humans or homo sapiens and the variation is found on the 12:40315247 chromosome. LRRK2 or leucine rich repeat kinase 2 is a gene that has variations linked to Parkinson's disease; LRRK2's functions are ATP binding, GTP binding, and GTP-dependent protein kinase activity. The disease-associated allele contains the GAG variation rather than the GTG codon, its numerical position is 40,315,266.
Primer Design and Testing
The non-disease primers came back from the test as chromosome 12:40315247 which makes sense because that was the one we were working on. It also concluded that the melting temperature for the non-disease forward primer was 44.7 C, and for the reverse it was 49.2 C. The disease primers that came back from the test returned zero results, and this is because the mutation does not allow it to read as chromosome 12:40315247. In the database that chromosome with a mutation does not exist as a primer.
BME 100 Fall 2018
