The Original Design
(Add image of the full OpenPCR machine here, from the Week 9 exercise. Write a paragraph description for visitors who have no idea what this is)
Experimenting With the Connections
When we unplugged (part 3) from (part 6), the machine's LCD display turned off.
When we unplugged the white wire that connects (part 6) to (part 2), the machine the temperature reading showed as -40 degrees Celsius and showed a wrong reading.
Test Run
On March 10th, 2014 between the hours of 9am and 12pm, the test run was attempted. 12 runs were completed and the machine passed.
Protocols
Thermal Cycler Program
DNA Sample Set-up
Positive Control- a
Negative Control- b
Patient 1 Sample 1- c
Patient 1 Sample 2- d
Patient 1 Sample 3- E
Patient 2 Sample 1- F
Patient 2 Sample 2- G
Patient 2 Sample 3- H
DNA Sample Set-up Procedure
Collect the following items: 8 empty PCR tubes, 8 PCR tubes filled with PCR liquid, the negative control, the positive control, and the 6 PCR tubes filled with samples from the patients.
Cut the PCR tubes so they are in groups of 4.
Label the side of the tubes with 8 different predetermined labels.
Put labeled tubes back in rack.
Using proper pipetting technique, transfer 50 μL of PCR reaction mix into each of the 8 labeled tubes.
In each of the labeled tubes, transfer the given DNA into the tube labeled for that DNA. Make sure to use a fresh pipette tip every single time!
Close all the lids tightly.
Take tubes to assigned PCR machine and wait for TAs to help,
PCR Reaction Mix
The PCR Reaction mix include Taq DNA polymerase, MgCl2, and dNTP’s.
DNA/ primer mix
Each DNA/primer mix includes the given DNA and forward/reverse primer.
Research and Development
PCR - The Underlying Technology
Finding the Disease-Associated Sequence:
In this lab we learned how PCR is used to detect a disease-associated DNA sequence(SNP). To do this we had to know a nucleotide is an organic molecule that serves as the monomer of nucleic acids like DNA and RNA. We also had to know polymorphism is two or more different phenotypes within the same species.
For this experiment we were given the SNP sequence rs237025. We found the species for this variation is homo sapien. This variation is found on teh chromosome 6:149721690 and the clinical significance was listed as other. This SNP is associated with the genes SUMO4 and TAB2. SUMO4, small ubiquitin-like modifier 4, is located in the cytoplasm of cells and specifically modifies IRBA. The diseases linked to it are type 1 Diabetes, Type 2 Diabetes, nephropathy, and VKH syndrome.
Designing a Disease Sequence-Specific Primer Pair:
An allele is and alternative form of a gene for a character producing different effects. The non-disease allele contains GTG, whereas the disease-associated allele contains the sequence ATG.
The numerical position of the SNP rs237025 is 149721690. The forward primer needed to design a diseased-allele-specific primer pair is CACCACTTAGTAAACTAATG. The numerical position 200 bases to the right of the disease SNP is 149721890, and the reverse primer for the SNP is CGTAAGAGTTAATCTTTTGA. If the template contains the non-disease allele PCR should not occur. This is due to the disease-specific primer and template not binding 1oo% to the non-disease allele because the non-disease allele had a sequence of GTG, when the disease-assocoated allele has a sequence of ATG.
(BONUS points: Use a program like Powerpoint, Word, Illustrator, Microsoft Paint, etc. to illustrate how primers bind to the cancer DNA template, and how Taq polymerases amplify the DNA. Screen-captures from the PCR video/ tutorial might be useful. Be sure to credit the sources if you borrow images.)
BME 100 Spring 2014
