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: 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
| Tube Label || PCR Reaction Sample || Patient ID
| G1 + || Positive control || none
| G1 - || Negative control || none
| G1 36189-1 || Patient 1, replicate 1 ||
| G1 36189-2 || Patient 1, replicate 2 ||
| G1 36189-3 || Patient 1, replicate 3 ||
| G1 28313-1 || Patient 2, replicate 1 ||
| G1 28313-2 || Patient 2, replicate 2 ||
| G1 28313-3 || Patient 2, replicate 3 ||
DNA Sample Set-up Procedure
- 1. The DNA samples are transferred to a PCR tube.
- 2. Using a pipette, 50µL of the DNA primers are then added to the DNA sample. The pipette is then discarded.
- 3. Next, 50µL of the PCR reaction mix is added to the sample.
- 4. Each sample will be labled as above to avoid confusion.
- 5. The samples are then added to the PCR machine and run according to the values below.
Research and Development
PCR - The Underlying Technology
| Template DNA: || This is what the entire reaction is derived from. The template DNA is essentially the blueprint from which the newly replicated DNA from the reaction comes from.
| Primers: || Primers are just a single strand of DNA it is relatively short and helps initiate the actual replication process in PCR.
| Taq Polymerase: || This is the enzyme used in the reaction that does the actually copying of DNA.
| Deoxyribonucleotides (dNTP’s): || These are the building blocks to the newly replicated DNA, the A’s T’s C’s and G’s.
| INITIAL STEP: 95°C for 3 minutes: || This gets the DNA ready to be split into 2 for replication.
| Denature at 95°C for 30 seconds: || This when the DNA will break apart, it splits down the middle of the double Helix.
| Anneal at 57°C for 30 seconds: || In the annealing process the primers will Bind with the separated DNA.
| Extend at 72°C for 30 seconds: || The DNA polymerase will activate in this time frame and it finds the primers to begin replication.
| FINAL STEP: 72°C for 3 minutes: || This is the part when the deoxyribonucleotide parts are used to replicate DNA.
| FINAL HOLD: 4°C: || Bringing the temp down this low helps finalize the annealing process.
| Adenine (A): || Thymine (T)
|| Thymine (T): || Adenine (A)
|| Cytosine (C): || Guanine (G)
|| Guanine (G): || Cytosine (C)
Q4: During Annealing, temperature is dropped to 57°C, allowing the primer to attach onto the separated DNA strands. Then the temperature is raised to 72°C which allows the DNA polymerase to attach to the primer. Next, the DNA polymerase attaches complementary nucleotide bases to the corresponding bases in the original strand of DNA.
SNP Information & Primer Design
Background: About the Disease SNP
SNP is a genetic disorder linked with Cardiac Arrhythmia. SNP stands for single-nucleotide polymorphism, a condition which arises from a mutation of a single nucleotide, in turn causing a change in the amino acid paired with the RNA codon during the translation phase of DNA replication. In the case of Cardiac Arrhythmia, it means that the nucleotide “C” is changed to an “A” in the sequence “CTC”. “CTC” in the DNA strand corresponds to the amino acid Glutamic Acid after transcription to RNA and during translation into the protein, however, when the sequence is changed to “ATC”, it reads as a stop codon, signaling the end of the production of the protein. This change in the structure of the protein causes an irregular heartbeat in patients.
Primer Design and Testing
The normal sequence of DNA was found successfully when we tested the non-disease primer, meaning we actually have the correct series of nucleotides identified, and the second failed because we used the allele of the gene rather than the normal version.
Non-Disease Primer Results:
Disease Primer Results: