LAB 4 WRITE-UP
- Lab coat and disposable gloves
- PCR reaction 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 and reverse primer.
- A strip of empty PCR tubes
- Cup for discarded tips
- OpenPCR machine: shared by two groups
PCR Reaction Sample List
| Tube Label || PCR Reaction Sample || Patient ID
| G6 + || Positive control || none
| G6 - || Negative control || none
| G6 1-1 || Patient 1, replicate 1 || 91356
| G6 1-2 || Patient 1, replicate 2 || 91356
| G6 1-3 || Patient 1, replicate 3 || 91356
| G6 2-1 || Patient 2, replicate 1 || 85189
| G6 2-2 || Patient 2, replicate 2 || 85189
| G6 2-3 || Patient 2, replicate 3 || 85189
DNA Sample Set-up Procedure
- Collect the materials listed above.
- Cut the empty PCR tubes strip to create two strips of 4 tubes.
- Label the sides of the empty tubes with the labels in the table (above).
- Place tubes in a rack.
- Using the empty tube labeled positive control, put 50 μL of PCR reaction mix using the micropipettor.
- Dispose of the used micropipettor tip in the cup.
- Using a new tip, place 50 μL of the positive control DNA/primer mix into the tube containing the correlating PCR reaction mix.
- Dispose of the used micropipettor tip.
- Repeat steps 5-8 for the negative control, patient 1 (replicates 1, 2, 3), and patient 2 (replicates 1, 2, 3), using the labeled tubes.
- Check to ensure that each finished tube has a total of 100 μL of PCR reaction mix.
- Close the lids tightly of the finished reaction tubes.
- Place the tubes into the OpenPCR machine, waiting until all 16 slots are filled with test tubes.
Let the thermal cycler lid heat to 100°C. Put the DNA samples into the thermal cycler at 95°C for 2 minutes. This is followed by 25 cycles of: denaturing the DNA at 95°C for 30 seconds, annealing at 57°C for 30 seconds, and extending at 72°C for 30 seconds. After the 25 cycles, let sit in 72°C for 2 minutes. Then let the thermal cycler sit at 4°C as a final hold.
Research and Development
PCR - The Underlying Technology
Q1. What is the function of each component of a PCR reaction?
| Template DNA ||
This is the target DNA sample that needs to be copied
| Primers|| These 2 proteins mark which section of the DNA sample needs to be amplified
| Taq Polymerase || This enzyme attaches to the primers and builds new DNA half-strands corresponding to the template DNA
| Deoxyribonucleotides || These are the basic blocks that are assembled to correspond to the template DNA
Q2. What happens to the components during each step of thermal cycling?
| INITIAL STEP: 95°C for 3 minutes: || Required during some variations of PCR. It is done before adding the polymerase, and is there to kick-start normally unreactive taq polymerase species
| Denature at 95°C for 30 seconds: || This hot temperature unwinds and separates the DNA strands into two half-strands
| Anneal at 57°C for 30 seconds: || The cooler temperatures allow the primers to bond to the unwound DNA half-strands, also preventing the 2 halves from rewinding
| Extend at 72°C for 30 seconds: || Raising the temperature allows the Taq Polymerase to begin assembling the new strands of DNA
| FINAL STEP: 72°C for 3 minutes: || In this final step the temperature is held at a higher temperature to allow any unfinished segments to finish assembling
| FINAL HOLD: 4°C: ||Bringing the temperature down and holding at this low temp maintains the integrity of the sample until it is ready to be removed.
DNA Nucleotides and their binding pair.
| Adenine (A):||T|| Thymine (T):||A|| Cytosine (C):||G|| Guanine (G):||C
Q4. During which two steps of thermal cycling does base-pairing occur?
Base-pairing occurs during the extending portions of the cycle and at the end to finish any unfinished base pairings. The higher temperatures of these steps prompt the taq polymerase to begin base pairing.
SNP Information & Primer Design
Background: About the Disease SNP
The disease SNP is representative of the disease cardiac arrhythmia syndrome, a disease in which the heartbeat of the patient is irregular. This includes fast, slow, and off-beat heartbeats. Arrhythmias are related to issues with the electrical conduction system of the heart. The condition can be treated effectively, utilizing pacemakers and other medication in conjunction with blood thinners. The disease can be found in any chamber of the heart and is treated in the same fashion.
Primer Design and Testing
The results of the primer test concluded that the primers that were designed were indeed compatible with the disease of cardiac arrhythmia. The results proved that the primers were a 220bp sequence from chromosome 4, which was the chromosome in question in regards to the disease.