Name: Jocelynn Christensen
Role: Experimental Protocol Planner
Name: Sam Zimmerman
Role: OpenPCR Machine Engineer and Vampire Hunter Extraordinaire
Name: Ryan Uchimura
Role: Experimental Protocol Planner
LAB 1 WRITE-UP
Initial Machine Testing
The Original Design
Experimenting With the Connections
When we unplugged the LCD screen from the circuit board, the machine stopped displaying information on the LCD screen.
When we unplugged the white wire that connects the circuit board to the heating block, the heating block would not heat up.
We first tested the OpenPCR machine on October 25, 2012. We ran the machine through a preprogrammed sample test run. At first we were not getting consistent temperature readings outputting to the LCD screen; however, after we rebooted the OpenPCR machine and ran the test again the machine worked perfectly.
Polymerase Chain Reaction
1. The Polymerase Chain Reaction or PCR works by singling out a single piece of DNA and then multiplying it so there's millions of copies of one strand of DNA. It's a step by step process that first occurs by heating up the DNA to 100°C in order to denature the hydrogen bonds between the two strands of DNA so that both sides of the DNA can be accesible to copy. After the strands are separated, specific primers are added to locate the section of DNA to be amplified. Next, the Taq DNA polymerase is added which actually copies the section of DNA desired and synthesizes the second half of each strand. After this there are only a few copies of the DNA which is why the machine then replicates more strands by repeating the process multiple times until there are millions of copies.
- 1. Collect DNA from patients
- 2. Heat Denaturation- the sample is heated to break the bonds between the strands
- 3. Primer Annealing- the solution cools and the DNA matches up to the primer.
- 4. Extension- The DNA replicates to produce millions of copies of the one strand of DNA .
3. The GoTaq master mix contains 400µM dATP, 400µM dGTP, 400µM dCTP, 400µM dTTP, and 3mM MgCl2
| Reagent || Volume
| Template DNA (20ng) || 0.2μL
| 10μM forward primer || 1.0μL
| 10μM reverse primer || 1.0μL
| GoTaq master mix || 50.0μL
| dH2O || 47.8μL
| Total Volume || 100.0μL
5. Out of the eight samples we ran the PCR on, one was a positive control with the cancer DNA template, the other was a Negative control with no cancer DNA template. Then we had three samples of a 57 year old male's DNA (patient ID 19185). We also had three samples of a 63 year old female's DNA (patient ID 88142).
(Add your work from Week 3, Part 2 here)
Research and Development
Specific Cancer Marker Detection - The Underlying Technology
- Our genes can tell us anything and everything about ourselves.
- The sooner we can detect cancer, the more effectively it can be prevented or treated.
- So why not find out about our disposition to cancer with our genes?
- The science community has identified many DNA sequences that are correlated to incidence of cancer. Through a process known as Polymerase Chain Reaction, (or PCR,) we can make tons of copies of any sequence of DNA from a DNA template. So, let's say we want to find out if someone has a sequence of DNA that may be indicative of a higher cancer risk; how can we do it?
r17879961 is a sequence of DNA that has been positively linked with cancer. It is a part of a sequence of DNA that codes for a protein kinase called CHEK2.
(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 OpenPCR tutorial might be useful. Be sure to credit the source if you borrow images.)
| Sample || Integrated Density || DNA μg/mL || Conclusion
| PCR: Negative Control || E6 || F6 || G6
| PCR: Positive Control || E7 || F7 || G7
| PCR: Patient 1 ID #####, rep 1 || E8 || F8 || G8
| PCR: Patient 1 ID #####, rep 2 || E9 || F9 || G9
| PCR: Patient 1 ID #####, rep 3 || E10 || F10 || G10
| PCR: Patient 2 ID #####, rep 1 || E11 || F11 || G11
| PCR: Patient 2 ID #####, rep 2 || E12 || F12 || G12
| PCR: Patient 2 ID #####, rep 3 || E13 || F13 || G13
- Sample = Comprised of two drops of DNA solution and two drops of SYBR Green.
- Integrated Density = The integrated density of the drop minus the integrated density of the background. This calculation account for background noise.
- DNA μg/mL = The integrated density of sample divided by the integrated density of drop multiplied by two.
- Conclusion = If the reading is positive then it means that the cancerous mutation is present.If the reading is negative or "no signal," then the mutation is not present.