LAB 1 WRITE-UP
Initial Machine Testing
The Original Design
Above is the OpenPCR machine used for the experiment. The PCR machine is a thermal cycler designed to precisely alter temperatures in order to begin the PCR reaction. It consists of a thermal block made out of aluminum where the sample containers are placed and heated/cooled. A lid with an adjustable seal covers the thermal block in order to maintain a constant temperature. In addition, a heat sink fan is mounted below to cool down the system when necessary. Software is used to adjust the number of cycles and temperatures desired for the experiment via USB/PC. A display screen let's the user know basic information corresponding to the system such as temperature, cycle number, and status of the machine.
Experimenting With the Connections
When we unplugged (part 3) from (part 6), the display turned off.
When we unplugged the white wire that connects (part 6) to (part 2), the display showed that the temperature changed from 24.6 to -40 degrees Celsius.
(We tested this machine on Wednesday, March 19th. The Open PCR machine ran smoothly and efficiently. It completed the 35 cycles within approximately one hour and forty minutes. We experienced no diffiuclties in the inital running.)
Thermal Cycler Program
DNA Sample Set-up
| Positive Control: Label: PC1 || Patient 1 Replicate 1: (1R1) ID: 56366 || Patient 1 Replicate 2: (1R2) ID: 56366 || Patient 1 Replicate 3: (1R3) ID: 56366
| Negative Control: Label: NC1 || Patient 2 Replicate 1: (2R1) ID: 86361 || Patient 2 Replicate 2: (2R2) ID: 86361 || Patient 2 Replicate 3: (2R3) ID: 86361
DNA Sample Set-up Procedure
- Obtain 8 empty attached PCR tubes and PCR reaction mix in 8 attached tubes, each with 50 microliters
- The 8 empty tubes are labeled with postiive control," "negative control," and all the patient ID's.
- Place tubes in the rack.
- Use proper pipetting technique to transfer 50 microliters of th ePCR reaction Mix to the postive control tube. Discard tip afterwards. Repeat for the rest of the 7 tubes.
- Add 50 microlioters of the "+" DNA/primer mix into the postiive control tube.
- Repeat process for negative control, and patient 1 replicates 1, 2, 3, and patient 2 replicates 1, 2, and 3. Use the appropriate DNA/primer mix for each of the corresponding tubes.
- Close the lids on all the OCR reaction tubes. Each one should have a total of 100 microliters.
- Place tubes in PCR machine and begin reaction when instructed to do so.
PCR Reaction Mix
- The PCR reaction mix contains Taq DNA polymerase, MgCl2, and dNTP's.
DNA/ primer mix
- What is in the DNA/ primer mix? All DNA/primer mixes contain the same forward primer and reverse primer, with a different template DNA
Research and Development
PCR - The Underlying Technology
Nucleotides are the four types of molecules, A, T, C, and G that form base-pairs. A polymorphism in genes can be a small-scale multi-base deletion or insertion of a different base in a code, a single-base nucleotide substitution, or retroposable element insertions and microsatellite repeat variations. Based on searching rs237025 in the database of short genetic variations in the NCBI website, we found that this variation is found in Homo Sapiens. The variation in the gene is located on chromosome 6:149721690. No specific clinical significance was listed. This SNP is associated with the SUM04 and TAB2 genes and is linked to type I diabetes.
The SUM04 gene associated with the SNP stands for a small ubiquitin-like modifier 4 which encodes small ubiquitin-related modifiers attached to proteins and can control the target proteins’ subcellular localization, stability, or activity. An allele is the alternate form of a gene that is caused by mutation and found in the same place on a chromosome. In this SNP, the non-disease allele contains GTG but a change in the first G position is linked to the disease. The disease-associated allele contains the sequence ATG.
The SNP is located at position 149721690. The disease-allele-specific forward primer ending with the nucleotide from the disease-associated allele is AACCACGGGGATTGTCAGTG. A PCR reaction needs two primers to amplify DNA so the position of the reverse primer 200 bases to the right of the disease SNP 149,721,890 and is AGTTTTCTAATTGAGAATGC.