LAB 1 WRITE-UP
Initial Machine Testing
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 ... (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)
(Write the date you first tested Open PCR and your experience(s) with the machine)
PCR, or Polymerase Chain Reaction, is a method of amplifying a particular segment of DNA by way of selective replication. If the sequence of the target DNA segment is known, primers can be created from the complimentary base pairs at each end of the target segment. A forward as well as a reverse primer is required so that each double strand of DNA results in two double strands of DNA at the end of each cycle (as Taq DNA Polymerase only works in one direction, it needs a primer at the beginning of both strands in order to copy both). To complete PCR, a DNA sample (containing the target DNA segment) must be mixed with the primers (both forward and reverse as discussed above), Taq DNA Polymerase, dNTP's (free base pairs to be used as the building blocks in the new DNA put together by the polymerase), and MgCl2 (a required substrate for the polymerase to use the dNTP's).
This solution is then subject to multiple cycles of varying temperatures. The temperature at each stage is chosen to optimize the desired activity of the various ingredients. First, the solution is heated to 95°C for 3 minutes to denature the double stranded DNA into two single strands. This makes the nucleotides available to attaching to the primers (which are much more abundant in the solution than the initial strands of DNA) when the solution is cooled to 57°C. After 30 seconds, the solution is brought up to 72°C, the temperature at which the activity of Taq DNA polymerase is optimal. This temperature is held for 30 seconds while Taq DNA polymerase attaches to the primers and replicates the DNA. This single cycle results in twice as many strands of double-stranded DNA as was put in. The cycle is repeated 35 times with each temperature being held for 30 seconds each. When sufficient copies of the DNA segment have been made, the temperature is decreased to 4°C to stop the reaction.
A step-by-step break down of the procedure follows.
Thermal Cycler Program
95°C for 3 minutes: Initial DNA strand is separated.
35 cycles of the following steps, each with a duration of 30 seconds:
- 95°C: Double strands of DNA separate.
- 57°C: Primers attach at ends of target DNA segment.
- 72°C: DNA polymerase activates and replicates target segment of DNA.
Final Hold 4°C for 3 minutes: PCR reaction is stopped.
DNA Sample Set-up
Two samples of DNA were tested in triplicate. Patient 1's ID number is 29013 and Patient 2's ID number is 13146. The samples for Patient 1 were labeled α1, α2, and α3. Similarly, Patient 2's samples were labeled β1, β2, and β3. Additionally, a positive control (CP) and a negative control (CN) were used. The reaction tubes containing the samples and PCR reaction mix were arranged for PCR reaction as follows:
DNA Sample Set-up Procedure
- 8 reaction tubes were provided to us, each containing 50μL of PCR reaction mix
- All reaction tubes were labeled according to the table above in order to avoid swapped results
- 50μL of each DNA sample Mix was added to the correspondingly labeled reaction tube (using a new pipette tip for each transfer in order to avoid cross-contamination between samples)
- The reaction tubes were placed into the thermocycler
- The thermocycler program detailed above was run so that PCR would occur in each reaction tube
PCR Reaction Mix
50μL of the following:
- Taq DNA polymerase
- forward primer
- reverse primer
50μL of the following:
- Extracted sample of a particular patient's DNA
Research and Development
Specific Cancer Marker Detection - The Underlying Technology
(Add a write-up of the information discussed in Week 9's class)
(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.)