BME103 s2013:T900 Group1

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Lab Write-Up 1
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Contents

OUR TEAM

Kristi Norris:Protocol Planner
Kristi Norris:
Protocol Planner
Carlos Renteria:Research and Design Specialist
Carlos Renteria:
Research and Design Specialist
Raul Monzolo:Open PCR Machine Engineer
Raul Monzolo:
Open PCR Machine Engineer
Johnny Montez:Open PCR Machine Engineer
Johnny Montez:
Open PCR Machine Engineer
Robert Sanchez:Research and Design Specialist
Robert Sanchez:
Research and Design Specialist
Group 1
Group 1

LAB 1 WRITE-UP

Initial Machine Testing


The Original Design
Image:Pcrphotoimage.JPG

The device displayed above is an polymerase chain reaction which is known as an "OpenPCR" is a biochemical technology which is used to amplify DNA. It works by having mutilpe cycles of repeated heating and cooling of the reaction for DNA melting and enzymatic replication of the DNA. The PCR is hooked up to a computer via usb which is how we control the number of cycles we wish it to repeat. Once the computer knows the desire amount of cycles to run through it is heated/cooled by the heating lead, heat sink, and fan. The circuit board than computes the information and transsfers it to the Lcd screen to show real time results.


Experimenting With the Connections

When we unplugged the LCD screen(part 3) from circuit board (part 6), the machine didn't display any information on the LCD screen.

When we unplugged the white wire that connects Circuit board (part 6) to heat block (part 2), the machine didn't read the temperatures accurately.

Image:ImagesCAMC1AOU.jpg‎

Test Run

(Write the date you first tested Open PCR and your experience(s) with the machine)




Protocols

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

Stage 1
95°C for 3 minutes: Initial DNA strand is separated.
Stage 2
35 cycles of the following steps, each with a duration of 30 seconds:

  1. 95°C: Double strands of DNA separate.
  2. 57°C: Primers attach at ends of target DNA segment.
  3. 72°C: DNA polymerase activates and replicates target segment of DNA.

Stage 3
Final Hold 4°C for 3 minutes: PCR reaction is stopped.

Screen shot of the Open PCR program detailed above 

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:

CP α1 α2 α3
CN β1 β2 β3

DNA Sample Set-up Procedure

  1. 8 reaction tubes were provided to us, each containing 50μL of PCR reaction mix
  2. All reaction tubes were labeled according to the table above in order to avoid swapped results
  3. 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)
  4. The reaction tubes were placed into the thermocycler
  5. The thermocycler program detailed above was run so that PCR would occur in each reaction tube

PCR Reaction Mix

  • Taq DNA polymerase
  • MgCl2
  • dNTP's

DNA Sample/Primer Mix

  • Extracted sample of a particular patient's DNA
  • Forward Primer
  • Reverse Primer



Research and Development

Specific Cancer Marker Detection - The Underlying Technology

Polymerase chain reaction (abbreviated PCR) is the process by which a selected strand or segment of DNA is replicated various times. Each step in the process is vital for the process to occur efficiently.

In the first step of PCR where the samples were heated to 95ºC for three minutes, the two strands of DNA were separated from one another due to the high amount of heat in the reaction. This temperature and the amount of time that the samples were heated were both necessary because they allowed for an adequate amount of energy necessary to break the hydrogen bonds between the parallel nucleotides. This caused the DNA sequence to unzip, which opens up the opportunity for the entire reaction to occur. After this occurred, 35 cycles of the samples being heated up to 95ºC, down to 57ºC, down to 72ºC occured. Again, at 95ºC the DNA was separated in order for the individual components to begin mixing together, like a soup. At 57ºC, the samples were cooled down, which allows for the forward and reverse primers to bind to their respective regions. When reheated to 72ºC, the TAQ polymerase was activated which generated a new strand of DNA from both the lagging and leading strands. The primers tell the polymerase where to begin generating new sequences of DNA, with the Magnesium Chloride acting as a catalyst for this reaction. At 72ºC, the temperature is adequate enough to where the polymerase can generate a new strand of DNA from the template quickly without denaturing. After this final step, the new sequences of DNA are fully generated and the process begins over again 35 times, each resulting in two new DNA strands.

This helps us because only the cancerous DNA is replicated, whereas the original template DNA is not replicated at all. This is because the primers are designed to bind only to the cancerous sequences of DNA. This is because that specific type of cancer's DNA sequence was analyzed and a primer generated that binds to that analyzed, cancerous sequence. As such, when the primers detect this, they bind to those, and the DNA polymerase begins generated a sequence of DNA for this strand. This does not occur in the normal strand of DNA, simply because it doesn't have that specific sequence, and as such the primers won't bind to it. The mutation in the cancer allows for those specific primers to bind to that sequence, but the opposing, normal strand will not bind to it, even when the strand is continuously copied because the mutation isn't present there. As such, the cancerous sequence will be replicated, while the normal strand remains uncopied.


Image:Sample DNA Example.png

This image shows exactly how the process works. In the beginning, at the top left, is the template DNA once it starts up. The second image down shows what occurs when the temperature is increased, then decreased. The template opens up allowing for each component of the PCR to go between the two strands, and once it's cooled down, the primers bind to each corresponding end of the DNA sequence that matches it. Keep in mind, that these primers were designed to bind only to the segments that have the corresponding cancer gene, otherwise it won't bind to them. What occurs next is when the temperature goes up, which is the next two images below. TAQ polymerase is activated, goes to the primer, and begins creating a segment of new DNA using the dNTPs that are floating around to create this DNA strand, as well as the Magnesium Chloride to act as a catalyst. This then creates the two new strands of DNA, and this continues, causing the DNA strand to grow exponentially over time. The image only shows what occurs with the cancerous DNA, however. The normal DNA strand is also replicated, but nowhere near as rapidly as the cancerous one due to the primers that are in the solution not matching up with that of the non-cancerous strand of DNA. It replicates at a non-exponential rate, leaving a far larger abundance of the cancerous DNA throughout the process.


(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.)





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