PCR reaction mix, 8 tubes, 50 μL each: Mix contains Taq DNA polymerase, MgCl 2, 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: only use each only once. Never reuse disposable pipette tips. If you do, the samples will become cross-contaminated
Cup for discarded tips
OpenPCR machine: shared by two groups
PCR Reaction Sample List
PCR Reaction Sample
Patient 1, replicate 1
Patient 1, replicate 2
Patient 1, replicate 3
Patient 2, replicate 1
Patient 2, replicate 2
Patient 2, replicate 3
DNA Sample Set-up Procedure
Label each of the 8 tubes with the names listed above
Pipette 50 µL of the PCR reaction mix into the positive control tube
Dispose of the pipette tip to avoid cross-contamination
Pipette 50 µL of the DNA/primer mix into the positive control tube (total volume in tube is now 100 µL)
Repeat steps 2-4 for the rest of the tubes with designated DNA/primer mix for each
Close the lids on tubes and insert them into thermal cycler
Heated Lid: 100 °C.
Initial Step: 95 °C for two 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 two minutes.
Final Hold: 4 °C.
Research and Development
PCR - The Underlying Technology
Components on a PCR Reaction
Template DNA: One strand of DNA used as a template to create a complementary strand. Because of this, one double stranded DNA molecule is converted into two, and both strands are identical to the first. When the DNA molecule is denatured in two single strands, each of this become the template for the PCR process.
Primers: Primers are short DNA or RNA sequence that mark the beginning point for DNA synthesis. Specifically, the bind to the 5-end of the template strand and Taq Polymerase binds to them to begin the DNA synthesis.
Taq Polymerase: The enzyme (protein) that assembles nucleotides into new strands of DNA.
Deoxyribonucleotides (dNTP’s): Single units of bases A, T, G, C
Components during each step of the thermal cycling
Initial Step: 95°C for 3 minutes:
Template DNA: Separates into two strands. Primers: The primers stay in the solution. They don’t interfere in the DNA molecules yet. Taq Polymerase: Not activated at this stage dNTP’s: The nucleotides bases stayed in the solution. They don’t interfere in the DNA molecules yet.
Anneal at 57C for 30 seconds:
Template DNA: The two single strands tend to go back together but the abundance of primers in the tube prevents them of doing this. Primers: Primers bind to the respective. complementary sequences in both single-strands at their 5-end. Taq Polymerase: Taq Pol is not activated at 57°C. dNTP’s: The nucleotides bases stayed in the solution. They don’t interfere in the DNA molecules yet.
Extend at 72°C for 30 seconds:
Template DNA: DNA is now bind by the primers and Taq Polymerase, ready to be synthesized. Primers: One Taq Polymerase protein binds to each primer. Taq Polymerase: One complex binds to the end of the primer. dNTP’s: Taq Polymerase begin to add them to the single-strands that are going to be synthesized.
Final Step: 72°C for 3 minutes:
Template DNA: Each single-strand is synthesized in a copy of the original double stranded DNA molecule. Primers: N/A Taq Polymerase: Adds the nucleotides creating the second complementary strand. dNTP’s: Nucleotides are added by Taq Polymerase matching respectively their complementary bases from the template single-strand.
Final Hold: 4°C:
Template DNA: There is now two DNA molecules for each original template DNA at each PCR cycle, and the synthesized molecules are stabilized at the low temperature. Primers: Remain in the solution if they were not the limiting reactant for the PCR. Taq Polymerase: Remain in the solution if they were not the limiting reactant for the PCR. process. dNTP’s: Remain in the solution if they were not the limiting reactant for the PCR.
Nucleotide Base Pairing
Adenine (A): Thymine (T).
Thymine (T): Adenine (A).
Cytosine (C): Guanine (G).
Guanine (G): Cytosine (C).
Base Pair Occurrence
Stage Occurrence: Base-pairing occurs at the annealing step when the primers bind to the DNA strand. It also occurs at the extension step, when the DNA polymerase adds complementary base pairs to the target strand.
Polymerase Chain Reaction Model:
SNP Information & Primer Design
Background: About the Disease SNP
SNP, which stands for “single nucleotide polymorphism”, is a disease that causes a genetic alteration common in many individuals. The disease occurs when there is a single change in the nucleotide, or a building block of DNA consisting of Adenine, Thymine, Guanine, and Cytosine. On average, SNP is located around every 300 nucleotides, which is a total of 10 million in the human genome. In our lab, we observed a variation of SNP located on the 7:117587799 chromosome. This specific SNP was linked to cystic fibrosis.
"What Are Single Nucleotide Polymorphisms?" U.S. National Library of Medicine. National Institutes of Health, 21 Mar. 2017. Web. 21 Mar. 2017.
Primer Design and Testing
For this lab, two sets of primers were designed, the non-disease primers and the disease-specific primers. The non-disease forward primer was written from the position of the SNP for a sequence of 20 bases long, and its reverse primary was written till the base that was 200 base pairs to the right of the SNP. Then, the disease primers were written by changing the final base of the non-disease forward primer to match the disease SNP nucleotide.
We ran two different tests with the non-disease and the disease-specific primers. From the tests, we observed that the non-disease primers resulted in a match with the same chromosome as the chromosome we found earlier, confirming our results. In addition, the disease primers produced no matches, since the diseased codon would mutate at the AGT sites.