BME100 f2013:W900 Group7 L4

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BME 100 Fall 2013 Home
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Lab Write-Up 1 | Lab Write-Up 2 | Lab Write-Up 3
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Contents

OUR TEAM

Name: Neel Akash PendyalaProtocols
Name: Neel Akash Pendyala
Protocols
Name: Abby MonhollenResearch and Development
Name: Abby Monhollen
Research and Development
Name: Christopher G. CusickMachine Testing
Name: Christopher G. Cusick
Machine Testing
Name: Shelby S. MartinProtocols
Name: Shelby S. Martin
Protocols
Name: Roberto AguirreMachine Testing
Name: Roberto Aguirre
Machine Testing

LAB 1 WRITE-UP

Initial Machine Testing

The Original Design
Image:PCRmachineG7.jpg

A Thermocycler or DNA Amplifier, also known as a PCR Machine, is used to create a vast quantity of a specific sequence of DNA from a cell via saliva, hair strands, etc. After heating up the DNA in order to melt, and disconnect the DNA into two tracts, primers, which are small strands of DNA containing 15-20 nucleotides, are used to adhere to a small portion of the single stranded DNA. Next, taq polymerase, which is an enzyme designed for DNA replication, replicates the rest of the DNA strand at a cooler temperature, creating a new doubled-stranded piece of DNA. After multiple cycles of this process, the PCR machine will have creating billions of replicated DNA strands.



Experimenting With the Connections

The insides of the PCR machine has a complex of wiring combined with essential components that make the machine work and if there are any faults or misconnections, unreliable data will be loaded and the test will be inaccurate. By unplugging the white wire that connects the circuit board to the heating plate, the LCD displayed an inaccurate temperature reading of -40.0 degrees Celsius. Once the white wire was plugged back into the right connection, the correct reading of 24.4 degrees Celsius was displayed and the temperature monitor was determined to be working properly. Each and every connection within the PCR Machine casing was checked thoroughly to ensure that the device will work the way it was intended. If any of these components, such as the heater, circuit board, fan, or any other critical part of the PCR machine, was not connected properly, inaccurate and false readings would be recorded and the test would be negative.

Image:PCRdiagramG7.png

Test Run

The initial test run of the device was on October 23, 2013 at 9:51 a.m. The machine was plugged into the school program through the USB port and the OpenPCR software was opened up. Using the software was simple and very straight-forward to use. The machine interfaced flawlessly with the computer software and began testing the device for 35 cycles. A an estimated completion time was displayed of one hour and forty-three minutes. Although this seemed a reasonable estimate, after fifteen minutes of testing, the estimated time read one hour and forty minutes. This was not a very accurate time estimate and after an hour of going through cycles, one hour was still displayed on the computer software. As far as the machine and its performance, it worked as it was intended. Each cycle went through the temperature changes specified and ran according to our specified parameters.




Protocols

Thermal Cycler Program

  • Initial Step: 95°C for 3 minutes
  • Middle Step: 35 cycles, denature at 95°C for 30 seconds, anneal at 57°C for 30 seconds, extend at 72°C for 30 seconds.

Denaturing: The double stranded DNA is "denatured" or separated into two single strands due to the breaking of hydrogen bonds between the complementary base pairs of the double-stranded DNA.

Annealing: Primers, single-stranded DNA sequences, attach to the ends of the target DNA sequence (foward primer attaches to one strand of the template DNA while reverse primer attaches to the other).

Extending: Taq DNA polymerase extends the primers, adding dNTPs and replicating the target DNA sequence.

  • Final Step: 72°C for 3 minutes
  • Final Hold:4°C

At this temperature, the DNA can neither be separated nor replicated and so the PCR reaction ends.

DNA Sample Set-up
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DNA Sample Set-up Procedure

  1. Collect all of the needed materials.
  2. Plug-in and turn on the Open PCR Machine, also make sure that the machine is ready for testing (see previous section for how to prepare the machine for testing).
  3. Pipet the eight individual DNA samples in to the respective tubes (see above table).*
  4. Pipet the PCR reaction mix in to each of the tubes.
  5. Pipet the DNA/primer mix in to each of the tubes.
  6. Set the tubes in the Open PCR Machine, make sure the lid is closed properly.
  7. Run Open PCR.
  8. Store tubes for later experimental use.
  9. Clean up all materials.
        *When pipetting, make sure to dispose of each pipet after one use. Do not cross contaminate samples by using the same pipet continuously. 

PCR Reaction Mix

  • What is in the PCR reaction mix?

There are 8 PCR reaction tubes, each of which contain a total of 100 μL of solution. The components of the PCR reaction mix, which make up half of the solution, are as follows:

  • Taq DNA polymerase- replicates the target DNA sequence by adding dNTPs that are complementary to those of the template strand
  • MgCl2- provides magnesium ions that bind to the dNTPs and allow the dNTPs to be used up by Taq DNA polymerase
  • dNTPs- added by Taq DNA polymerase to elongate the primers, thereby forming strands of DNA

DNA/ primer mix

  • What is in the DNA/ primer mix?

The other half of the solution in each tube consists of the following components:

  • Sample of DNA from a patient that serves as the template DNA in the PCR reaction
  • Forward and reverse primers- bind to template strands and initiate replication




Research and Development

PCR - The Underlying Technology

PCR Components:

The template DNA is used in the polymerase chain reaction (PCR) as a model for DNA copying. The template DNA contains the sequence of information that is to be amplified through PCR and from the template DNA comes the numerous copies of the sequence. In PCR, primers are used to "mark" the starting point for the target sequence. A primer attaches to a specific sequence of bases at the beginning of a target sequence on each single strand of DNA (for a total of two primers per double strand of DNA). The primers then allow taq polymerase to add complementary bases. The taq polymerase attaches to the primer because it must add bases to an existing piece of DNA. After attaching to the primer, the taq polymerase adds nitrogen bases according to base pairing rules, creating a second strand of DNA. Magnesium chloride catalyzes this process, allowing the taq polymerase to bind to the primer faster and easier that it would under standard conditions. The deoxyribonucleotides (dNTP's) are bases in the mixture that are added to the new strand of DNA by the taq polymerase.

Base Pairing Rules:

Taq polymerase adds bases to the template DNA strand according to the following base pairing rules: Adenine (A) is paired with Thymine (T) and Thymine is paired with Adenine, Guanine (G) is paired with Cytosine (C) and Cytosine is paired with Guanine

PCR Reaction Steps: First, the reaction mixture is brought to a temperature of 95 degrees Celsius for three minutes. This causes the template DNA to denature. The mixture remains at this temperature for an additional thirty seconds, which allows the double helix of the DNA to separate into two single strands of DNA (Denaturing). This happens because the heat causes the hydrogen bonds between the complementary base pairs to break. Next, the mixture is cooled to 57 degrees Celsius for thirty seconds. At this temperature, primers lock onto their target sequence. This process is known as annealing. After the primers lock onto their target sequence, the temperature is raised to 72 degrees Celsius for thirty seconds, which causes the taq polymerase to activate. The taq polymerase locates an attached primer, where it begins adding complementary bases. The taq polymerase continues this process, extending the complementary strand of DNA, until it reaches the end of the template DNA. The mixture then remains at 72 degrees Celsius for an additional three minutes, where the target DNA fragments are replicated to create billions of copies. After this process is complete, a final hold is done at four degrees Celsius. This final hold stops the PCR process by preventing the taq polymerase from attaching. The DNA copies that were created are stabilized through this final hold.


primers

First, the primers bind to a specific region of the DNA that contains the cancer mutations. This allows PCR to replicate only a certain section of the DNA that contains the cancer gene/segment.

binding Taq polymerase then bonds to the primer that recently attached to a segment of DNA. The taq polymerase then duplicates the strands of DNA, making many copies of the particular segment under question, as shown below.

extending

Image Source: http://openpcr.org/what-is-pcr



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