BME103:T130 Group 6

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Contents

OUR TEAM

Name: Jocelynn ChristensenRole: Experimental Protocol Planner
Name: Jocelynn Christensen
Role: Experimental Protocol Planner
Name: Sam ZimmermanRole: OpenPCR Machine Engineer
Name: Sam Zimmerman
Role: OpenPCR Machine Engineer
Name: Adam HellandR&D
Name: Adam Helland
R&D
Name: Ryan UchimuraRole: Experimental Protocol Planner
Name: Ryan Uchimura
Role: Experimental Protocol Planner
Name: Dakota StyckRole: OpenPCR Machine Engineer
Name: Dakota Styck
Role: OpenPCR Machine Engineer

LAB 1 WRITE-UP

Initial Machine Testing

The Original Design

Image:Open PCR Solid Works Design.png

Image:BME 103 PCR SW.png

The images above depict our OpenPCR machine. It performs polymerase chain reactions (PCR), a process in which a particular DNA sequence is amplified. The DNA sequence can then be easily analyzed. The OpenPCR machine accomplishes the amplification of a DNA sequence through a series of heating and cooling sequences.


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.


Test Run

We first tested our OpenPCR machine (machine #6) 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.




Protocols

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.

2.

    Procedure
  • 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



4.
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).


Flourimeter Measurements




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.)




Results

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


KEY

  • 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.


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