OUR TEAM
LAB 4 WRITE-UP
Protocol
Materials
- Lab coat
- Disposable gloves
- PCR reaction mix, 8 tubes, 50µL each: Mix contains Taq DNA polymerase, MgCl2, and dNTP's
- DNA/primer mix, 8 tubes, 50µL 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: one-time use only
- Cup for discarded tips
- Micropipettor
- 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 |
22171
|
| G6 1-2 |
Patient 1, replicate 2 |
22171
|
| G6 1-3 |
Patient 1, replicate 3 |
22171
|
| G6 2-1 |
Patient 2, replicate 1 |
64572
|
| G6 2-2 |
Patient 2, replicate 2 |
64572
|
| G6 2-3 |
Patient 2, replicate 3 |
64572
|
DNA Sample Set-up Procedure
- Label 8 empty PCR tubes on the side (NOT the cap) with the 8 sample label names. This includes a positive control (G6 +), a negative control (G6 -), 3 replicates of the Patient 1 sample (G6 1-1, G6 1-2, G6 1-3), and 3 replicates of (G6 2-1, G6 2-2, G6 2-3).
- Place the 8 labeled tubes in a rack.
- Use the micropipette to deposit 50µL of the PCR reaction mix into the empty tube labeled G6 +.
- Using a new micropipette tip, add 50µL of DNA/Primer mix containing the positive control DNA, for a total of 100µL in the tube.
- Repeat steps 3-4 for the negative control, 3 replicates of Patient 1, and 3 replicates of Patient 2 until each tube has 50µL of PCR mix and 50µL of DNA/Primer mix. Be sure to use the DNA/Primer mix corresponding to the correctly labeled tube. Be sure to replace the micropipette tip between every use to avoid cross-contamination.
- Securely close the lid on each of the eight tubes.
- Bring the tubes to the PCR machine and wait until a total of 16 tubes are inserted into the machine before running PCR.
OpenPCR program
- The heated lid begins at 100ºC.
- For the initial step, the samples are held at 95ºC for 2 minutes.
- 25 cycles are completed in which the samples are denatured for 30 seconds at 95ºC, annealed for 30 seconds at 57ºC, then extended for 30 seconds at 72ºC.
- The final step holds the sample tubes at 72ºC for two minutes.
- The final holding temperature is 4ºC.
Research and Development
PCR - The Underlying Technology
Q1) Functions of Components of PCR
- 1. Template DNA is the original DNA that is being copied in the PCR process and contains the target sequence for copying. The template DNA begins being copied as it is heated up to near boiling until the double stranded DNA is split into two single strands.
- 2. Primers are short sections of DNA that are custom built in a lab with any combination of nucleotides that the creator wishes. Primers are used in DNA replication by having two attach to one strand at different section on the strand. Since DNA polymerase cannot attach specific place on the DNA strand, the primers act as barriers for the DNA polymerase to attach and add nucleotides.
- 3. Taq polymerase is a set of proteins that copy the DNA of a cell. DNA polymerase copies a cell’s DNA by attaching to one end of a primer (which is attached to a single strand of DNA) and begins adding nucleotides towards the other primer. The space between the two primers is now filled with a copy of the template DNA.
- 4. Deoxyribonucleotides (dNTP’s) are the simple components that make up DNA molecules. They consist of four types of nucleotides, Adenine, Cytosine, Guanine, Thymine. In DNA replication, the Taq polymerase grabs nucleotide floating in the liquid and attaches them to the end of the primer.
Q2) What happens to the components during each step of thermal cycling?
- INITIAL STEP: THis heats up the DNA, preparing it for a process known as denaturing.
- DENATURE at 95°C for 30 seconds: Denaturing causes it to completely separate from each other.
- ANNEAL at 57°C for 30 seconds:Primers attach to target sites before the DNA can reattach.
- EXTEND at 72°C for 30 seconds:At this step is where TAG polymerase is activated and finds the primers
- FINAL STEP: 70°C for 3 minutes: This is where the tag polymerase begins adding deoxyribonucleotides starting at the primer
- FINAL HOLD: 4°C: This is where it comes to a stop
Q3) Base Pairing
- Adenine(A): - Thymine
- Thymine(T): - Adenine
- Cytosine(C): - Guanine
- Guanina(G): - Cytosine
Q4) During which two steps of thermal cycling does base-pairing occur?
The base-pairing occurs between the steps of Annealing and Extension. In the annealing step, the system is cooled to 57*C and the target DNA is binded with short DNA primers that serve as starting positions for replication. Next, the temperature is raised to 72*C, enabling the DNA Polymerase to attach to the primer. Finally, the DNA polymerase expedites the corresponding nucleotide bases to the bases in the original DNA strand. All in all, Annealing and Extension together allow for base-pairing to occur.
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SNP Information & Primer Design
Background: About the Disease SNP
An SNP or Single Nucleotide Polymorphism is a variation in a person's genes. They also help when searching for diseases in a person's genome. The forward and reverse primers can help find the disease carrying base in someone’s gene sequence. Cystic Fibrosis was the condition linked to this particular SNP. The disease associated allele is CGT while the non-diseased allele is AGT. This shows that the C base is connected to the disease and that the only thing necessary for the primer to become disease associated is for the last base of the primer to change from A to C. An SNP can occur one in every few hundred bases. In this case this position was 200 spaces away and showed the diseased associated allele.
Primer Design and Testing
The non-diseased primer has a result of 220 bp which means that there are 220 bases in between the pair. The capital letters show the sequence matching the database and the lowercase letters show where it does not. The reason that the diseased primer has no matches is because it did not exist in this particular gene sequence.
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BME 100 Spring 2017
