LAB 4 WRITE-UP
- Lab coat and disposable gloves
- PCR reaction mix (mix contains Taq DNA polymerase, MgCl2, and dNTP's
- 8 tubes, 50μL each
- A strip of empty PCR tubes
- Disposable pipette tips: only use each once. Never re-use disposable pipette tips or samples will be cross-contaminated
- Cup for discarded tips
- OpenPC machine: shared by two groups
PCR Reaction Sample List
| Tube Label || PCR Reaction Sample || Patient ID
| G16 + || Positive control || none
| G16 - || Negative control || none
| G16 1-1 || Patient 1, replicate 1 || 45421
| G16 1-2 || Patient 1, replicate 2 || 45421
| G16 1-3 || Patient 1, replicate 3 || 45421
| G16 2-1 || Patient 2, replicate 1 || 53807
| G16 2-2 || Patient 2, replicate 2 || 53807
| G16 2-3 || Patient 2, replicate 3 || 53807
DNA Sample Set-up Procedure
- Step 1 Heat lid to 100°C
- Step 2 Place the DNA/primer mix in the PCR tube
- Step 3 Heat the tube to 95°C for 2 minutes
- Step 4 Then cycle through 25 cycles of 95°C for 30 seconds, 57°C for 30 seconds, and 72°C for 30 seconds. Those steps allow the DNA to break, the primers to join, and then the DNA to bind respectively.
- Step 5 At last leave its at 72°C for 2 minutes
- Step 6 Hold your DNA at 4°C
Research and Development
PCR - The Underlying Technology
Q1. What is the function of each component of a PCR reaction?
- Template DNA: the sample DNA that contains the target sequence. At the beginning of the reaction, high temperature is applied to the original double-stranded DNA molecule to separate the strands from each other.
- Primers: short pieces of single-stranded DNA that are complementary to the target sequence. The polymerase begins synthesizing new DNA from the end of the primer.
- Taq Polymerase: a type of enzyme that synthesizes new strands of DNA complementary to the target sequence. It is the first and most commonly used of these enzymes.
- Deoxyribonucleotides(dNTP’s): single units of the bases A, T, G, and C, which are essentially "building blocks" for new DNA strands.
Q2. What happens to the components (listed above) during each step of thermal cycling?
INITIAL STEP: 95°C for 3 minutes: The contents of the PCR tube are heated up to 95°C in preparation for the steps that follow. The hot temperature is used to activate the thermostable polymerases that are used in the PCR reaction.
- Denature at 95°C for 30 seconds: During this step the DNA strands denature, meaning the hydrogen bonds between the bases are broken and the DNA is split into two single strands, ready for replication.
- Anneal at 57°C for 30 seconds: During this step the 2 primers bind to the different single DNA strands.
- Extend at 72°C for 30 seconds: During this step the taq polymerase locates a primer attached to a single DNA strand and begins to add the complementary nucleotides onto the strand.
- FINAL STEP: 72°C for 3 minutes: During this step the new double helix DNA strands are created by the taq polymerase as it creates complementary base pairs between the nucleotides on the DNA strand and the free nucleotides. It is kept at 72°C because this is the optimum temperature for the activity of taq polymerase.
- FINAL HOLD: 4°C: During this step all of the contents of the tube are inactive. Its main purpose is for short term storage of the new DNA strands.
Q3. Base Pairing
- Adenine (A): Thymine (T)
- Thymine (T): Adenine (A)
- Cytosine (C): Guanine (G)
- Guanine (G): Cytosine (C)
Q4. During which steps of thermal cycling does base-pairing occur?
- Annealing is one of the steps of thermal cycling where base-pairing occurs. This is where the DNA primers attach to the two stands of DNA and base-pairing occurs to the matching sequence of the template DNA.
- Extension is the other step of thermal cycling where base-pairing occurs. This is where the Taq polymerase bring the nucleotides. This polymerase attach to the primers and extend them and pair them in the order of the DNA sequence.
SNP Information & Primer Design
Background: About the Disease SNP
SNP or Single Nucleotide Polymorphism is a DNA sequence variation that occurs in about 1% of a population. SNP is when a single nucleotide (A, T, C, G) differs between members of a biological species or paired chromosomes. The SNP variation is found in Homo sapiens and is located on chromosome 16. The clinical significance of SNP is that it is pathogenic. SNP is associated with the MC1R gene; MC1R stands for Melanocortin 1 Receptor. The MC1R gene, which is linked to fair skin and red hair color makes you more susceptible to the disease Melanoma.
Primer Design and Testing
After determining the primers and testing them, the results confirmed that we identified the correct non-disease forward and reverse primers. It also confirmed we identified the proper disease primers because when entered, "no matches" was returned. This is because there is an error (the mutation) in the primer and therefore it can not be sequenced.