Name: Mayuri Gupta Role: Open PCR machine Engineer
LAB 1 WRITE-UP
Initial Machine Testing
The Original Design
Experimenting With the Connections
When we unplugged (part 3) from (part 6), the machine ... (did what? fill in your answer)
When we unplugged the white wire that connects (part 6) to (part 2), the machine ... (did what? fill in your answer)
Test Run
(Write the date you first tested Open PCR and your experience(s) with the machine)
Protocols
Polymerase Chain Reaction
Polymerase chain reaction is basically molecular photocopying and the process or technique used to make copies of small segments of DNA because it only targets specific segments of the DNA and that's what makes it useful. PCR works by mixing two DNA fragments, also known as primers which are about 20 bases long. The mixture is then heated and denatured and then the primers bind to their complementary sequences on the separated strands. Then, the polymerase extends primers into new complementary strands and it goes through about 30 cycles. PCR products are useful and can be used in many experiments like DNA fingerprinting and detection of viruses.
Receive three replicate DNA samples from two patients
Create a new program on the Open PCR system
Create three stages
Stage 1: 1 cycle, 95°C for 3 minutes
Stage 2: 30 cycles for 95°C for 30 seconds, 57°C for 30 seconds, 72°C for 30 seconds
Stage 3. 72°C for 3 minutes
Final hold: 4°C
Reagent
Volume
Template DNA (20 ng)
10.2 μL
10 μM reverse primer
1.0 μL
dH2O
47.8 μL
0 μM forward primer
1.0 μL
GoTaq master mix
50.0 μL
Total Volume
100.0 μL
The eight samples we ran in PCR were:
Patient ID, Gender, Ages
27762, F, 52
59448, F, 45
PCR steps of amplification
Flourimeter Measurements
(Add your work from Week 3, Part 2 here aka, steps of assembly to the flourimeter)
Research and Development
Specific Cancer Marker Detection - The Underlying Technology
There is a genetic relation to having cancer or not when an individual is over the age of 40. The specific gene, in this case, is located on chromosome 22, r17879961. To test an human's DNA for this cancer gene, we have go through a series of reactions called PCR on the DNA for replication and amplification of the patient's DNA strand.
As previously explained in PCR, a primer is needed for the DNA replication. Two primers that have to be created. One at the "completing" strand of the double strand, and one as the cancer detecting strand. On chromosome 22, the primer to detect r17879961 defect has to have the changed nucleotide. In this particular instance, it is a adenine replaced by the cancerous-related nulceotide cytosine. The primer will only bind to the matching sequence (testing sequence) because of the A-T, and C-G pairings. Since an A has been replaced with a C, the primer can't bind to the DNA strand beyond that specific nucleotide in the sequence. Due to the open double helix, further steps in the reaction will not happen, and no amplification will happen. No amplification means no visible results and the test will run negative.
If the cancer gene is present, then the matching primer will completely bind to the DNA strand. When this happens, the amplification sequence will be able to precede, and this will show up as a positive result.
(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
(Your group will add the results of your Fluorimeter measurements from Week 4 here)
BME 103 Fall 2012
