PCR reaction mix (8 50μL tubes with Taq DNA polymerase, MgCl2, and dNTP’s)
DNA/Primer mix (8 50μL tubes with different template DNA, and the same forward and reverse primers)
Strip of empty PCR tubes
Disposable pipette tips
Cup for discarded tips
micropipettor
OpenPCR machine
PCR Reaction Sample List
Tube Label
PCR Reaction Sample
Patient ID
G 10 +
Positive control
none
G 10 -
Negative control
none
G 10 1-1
Patient 1, replicate 1
99518
G 10 1-2
Patient 1, replicate 2
99518
G 10 1-3
Patient 1, replicate 3
99518
G 10 2-1
Patient 2, replicate 1
27737
G 10 2-2
Patient 2, replicate 2
27737
G 10 2-3
Patient 2, replicate 3
27737
DNA Sample Set-up Procedure
Gather the materials.
Because the OpenPCR machine does not hold all the tubes together, the strip of PCR tubes needs to be cut in half.
Using a black marker, label the sides of the PCR tubes with the following labels: G10 +A, G10 -B, G10 1-1, G10 1-2, G10 1-3, G10 2-1, G10 2-2, G10 2-3.
Place the tubes on a rack.
Starting with the empty tube labeled as the positive control. Using the pipette and proper technique, transfer 50 μL of PCR reaction mixture into the test tube.
Using a new, clean pipette tip, transfer 50 μL of DNA/primer mixture into the same tube. The total volume in the positive control PCR test tube should be 100 μL.
Repeat steps 5 and 6 for the rest of the PCR tubes. Use the appropriate DNA/primer mixture for the rest of the tubes. When completed, all of the test tubes should contain 100 μL.
Close the lids on the PCR tubes.
Place the tubes into the PCR machine and ensure that all 16 slots of the machine are filled.
OpenPCR program
Heated lid: 100 C
Initial step: 95 C for 2 minutes
Number of cycles: 25...
Denature at 95 C for 30 seconds, Anneal at 57 C for 30 seconds, and extend at 72 C for 30 seconds
Final step: 72 C for 2 minutes
Final hold: 4 C
Thermal cycling program - The steps of a thermal cycling program include denaturing, annealing, and extending. The denaturing process occurs at 95 C for 2 minutes to turn it into a single strand. The next step, annealing, 2 primers are used to bind the targeted strand. The final step, extension, is used to greatly increase the quantity of DNA.
Research and Development
Function
Polymerase Chain Reaction consists of four components: Template DNA, Primers, Taq Polymerase, and deoxyribonucleotides (dNTP’s). Each component has a major role in completing PCR. The template DNA provides the original DNA that contains the target sequence. It is the reference strand that is used to determine the formation and composition of the complementary strand. Primers are short pieces of single stranded DNA that are complementary to the target sequence. PCR begins by synthesizing new DNA from the end of the primer. The primer functions as a marker for Polymerase to bind to. The Taq Polymerase is a an enzyme that builds new strands of DNA complementary to the target/template DNA strand. Taq Polymerase is unique because it is able to add new base pairs and remain heat resistant. This is important because during PCR, a temperature cycle is constructed, and if the Polymerase is not able to tolerate the heating and cooling, PCR will not be able to function. dNTP’s supply the individual DNA bases: A, T, G, and C, which are the building for strands. Without dNTP’s, Taq Polymerase wouldn’t be able to add the base pairs. dNTP’s provide the base pairs for Polymerase to add to synthesize the new DNA strand.
Thermal Cycling
There are six steps for thermal cycling. The initial step is when the mixture is heated to 95°C for 3 minutes. During this step, the template DNA is being heated and prepped for denaturing. It will take 3 minutes for the DNA mixture to reach the next step. After the DNA mixture has been prepped, denaturing will take place for about 30 seconds 95°C. Weak hydrogen bonds are broken in the template DNA, and the DNA converts from a double-stranded helix to single strands. Next, the DNA mixture is annealed at 57°C for 30 seconds. The DNA mixture is cooled, which allows primers to bind to the complementary sequence. Next, the DNA is extended at 72°C for 30 seconds. At this stage, the optimal temperature for Taq Polymerase to extend the primers by adding nucleotides. dNTP’s provide Taq Polymerase for the individual base pairs that create the nucleotide. In the final step the DNA mixture is set to 95°C for 3 minutes. The provides more time to ensure that DNA has completed extending and the complementary strand has been created. In the final hold, DNA mixture is at 4°C to cool the DNA mixture and preserve the complementary strand.
Basepairing
There are four DNA nucleotides that are linked together in a DNA double helix. The molecules, called nucleotides, are designated as A, T, C, and G. Basepairing, driven by hydrogen bonding, allows base pairs to stick together. Adenine (A) anneals to Thymine (T). Cytosine (C) anneals to Guanine (G). During extending and the final step is when basepairing occurs. At 72°C, Taq Polymerase works at the optimum capability. Polymerase extends the primers and adds the nucleotides to create the new complementary strand. Once extending and final step is complete, Taq Polymerase has added the base pairs and the complementary strand is reunited with the original DNA strand.
SNP Information & Primer Design
Background: About the Disease SNP
The SNP rs36686 is found in Homo sapiens on chromosome 19. The gene associated with this SNP is UDP-GlcNAc:betaGal beta-1,3-N-acetylglucosaminyltransferase 3, of B3GNT3. This SNP can affect immune and inflammation genes and increase risk of non-Hodgkin lymphoma.
Primer Design and Testing
The primer pairs for this DNA sequence were found by analyzing the DNA in specific areas on the gene. The non-disease forward primer is 5'-GTGCGGGCTCCATCGCAACG. The non-disease reverse primer is 5'-GGAGGAAGGTGTCGCCCCTT. The disease forward primer is 5'-GTGCGGGCTCCATCGCAACA. The disease reverse primer is 5'-GGAGGAAGGTGTCGCCCCTT. The non-disease primers were entered into the UCSC InSilico PCR website and the result was a 220 bp sequence from chromosome 19. The disease primers were also run through the UCSC InSilico PCR website and no matches were found.
BME 100 Spring 2016
