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 primer and reverse primer
- A strip of empty PCR tubes
- Disposable pipette tips: only use each only once. Never re-use disposable pipette tips or
samples will be cross-contaminated
- Cup for discarded tips
- OpenPCR machine: shared by two groups
PCR Reaction Sample List
| Tube Label || PCR Reaction Sample || Patient ID
| G14 + || Positive control || none
| G14 - || Negative control || none
| G14 1-1 || Patient 1, replicate 1 || 69477
| G14 1-2 || Patient 1, replicate 2 || 69477
| G14 1-3 || Patient 1, replicate 3 || 69477
| G14 2-1 || Patient 2, replicate 1 || 37835
| G14 2-2 || Patient 2, replicate 2 || 37835
| G14 2-3 || Patient 2, replicate 3 || 37835
DNA Sample Set-up Procedure
- Use the PCR tube to hold the extracted DNA.
- Add Primer 1 to the PCR tube.
- Now add Primer 2 to the PCR tube.
- Add nucleotides to the PCR tube.
- Add DNA polymerase to the PCR tube.
- Place in the DNA thermal cycler.
- Heat solution at 95C for two minutes to allow the hydrogen bonds between the DNA strands to break, exposing the DNA strands.
- Primer annealing: The mixture is cooled to 57C for 30 seconds. This allows the primers to bind to their complementary sequence in the template DNA.
- Extension: The reaction is then heated to 72C for 30 seconds, the optimal temperature for DNA polymerase to act. DNA polymerase then extends the primers, adding nucleotides onto the primer, using the target DNA as a template.
- Repeat the previous steps 20-30 times, but only heat at 95C for 30 seconds instead of 2 minutes.
- At the end, cool and hold the solution at 4C.
Research and Development
PCR - The Underlying Technology
| Template DNA || A molecule of a nucleic acid, such as DNA, that serves as a pattern for the
synthesis of a macromolecule, as of RNA.
| Primers || Short pieces of DNA that are made in a laboratory. Since they're custom built,
primers can have any sequence of nucleotides you'd like.
| Taq Polymerase || A thermostable DNA polymerase and is frequently used in PCR. Taq
polymerase is a bacteria that lives in hot environments and is stated as an enzyme that can
withstand the protein-denaturing conditions for successful PCR.
| dNTP’s|| A nucleotide containing deoxyribose that is a constituent of DNA.
| INITIAL STEP: 95°C for 3 minutes: || Getting the reaction to a temperature of 95°C to prepare for DNA translation.
| Denature at 95°C for 30 seconds: || Causes the DNA to separate by breaking the hydrogen bonds between complementary bases, yielding single strands.
| Anneal at 57°C for 30 seconds: || The reaction temperate is lowered to 57°C allowing annealing of the primers to the single strand DNA template.
| Extend at 72°C for 30 seconds: || The DNA polymerase synthesizes a new DNA strand complementary to the template.
| FINAL STEP: 72°C for 3 minutes: || To ensure that any remaining single stranded DNA is fully extended.
| FINAL HOLD: 4°C: || This ensures short term storage of the reaction.
Q3) DNA is made up of four types of molecules called nucleotides, designated as A, T, C and G.
Base-pairing, driven by hydrogen bonding, allows base pairs to stick together
| Adenine (A): || || Thymine (T)
| Thymine (T): || || Adenine (A)
| Cytosine (C): || || Guanine (G)
| Guanine (G): || || Cytosine (C)
Q4) Phase 4 and 5 is when base-pairing is occurring. In phase 4 at 72 degrees Celsius DNA polymerase synthesizes a new DNA strand complimentary to the DNA template strand created earlier. During phase 5, any remaining single stranded DNA is still being fully extended.
SNP Information & Primer Design
Background: About the Disease SNP
The disease is known as MC1R melanocortin 1 receptor or alpha melanocyte stimulating hormone receptor. It changed by only one allele the C is changed to a T. This single nucleotide change is the cause of the mutation in the gene. Its location on the gene is 89,917,879 until 89,920,977. Its total length is 3,099 polynucleotides long.
Primer Design and Testing
For this experiment we had to find a forward non-disease primer, a non-disease reverse primer, a disease forward primer, and a disease reverse primer. The disease primers contains the single base that was changed which causes the mutation, and the non-disease primer does not. When you enter the data on the genome database, when you enter the non-disease primers it comes up with a chromosome sequence that we found earlier in the experiment. When you enter the disease primers then it comes up with nothing.