BME100 s2014:T Group2 L4

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Lab Write-Up 1 | Lab Write-Up 2 | Lab Write-Up 3
Lab Write-Up 4 | Lab Write-Up 5 | Lab Write-Up 6
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Contents

OUR TEAM

Name:Ekanem-Essang Akpan studentRole(s)
Name:Ekanem-Essang Akpan student
Role(s)
Name:Bateer Song studentRole(s)
Name:Bateer Song student
Role(s)
Name:Alexander Jones studentRole(s)
Name:Alexander Jones student
Role(s)
Name:Hannah Churchill studentRole(s)
Name:Hannah Churchill student
Role(s)
Name:Daylin Morgan studentRole(s)
Name:Daylin Morgan student
Role(s)
Name:Group 2
Name:Group 2

LAB 1 WRITE-UP

Initial Machine Testing

The Original Design

Image:OpenPCR2resize.jpg

The image above represents a fully assembled OpenPCR machine. This device is used in conjunction with a computer to facilitate a polymerase chain reaction. A PCR uses heat and a specific mix of primers, DNA, nucleotides, and DNA polymerase. The way an OpenPCR strings together reactions is through heating the DNA to separate the strand. Then cooling slightly to allow the primers to attach to DNA. After the DNA polymerase finds the primers and adds complementary nucleotides, the PCR machine heats the tubes up again to begin the next cycle. This can be repeated more than 30 times.

Experimenting With the Connections

To fully understand the OpenPCR machine it was slightly disassembled. When the LCD Screen, part 3, of the diagram below was unplugged from the Circuit Board, part 6, the LCD screen turned off. When part 6 was unplugged from 16-tube well, part 2 the first temperature on the LCD screen was changed to reflect an inaccurate measurement. Both of these wires and all another connections withing the device are imperative to insure the PCR occurs without any errors.


Image:OPENPCRLABELS.jpg

Test Run


The initial OpenPCR test was ran on 3/20/2014. The results of this test were unsatisfactory since the device performed its operations too slowly. For the purposes of this lab the cycles should have been completed after closer to two hours. Since the machine was taking longer than should have been normal, the test was stopped and concluded as having failed. When using the OpenPCR it is vital to monitor its progress both on the computer application and the device itself to make sure that the reaction is progressing as it should. So time is not wasted on a failed PCR.




Protocols

Thermal Cycler Program
For the program, the steps used are as follows;
Stage one:
1 cycle at 95 degrees Celsius for 3 minutes. Initial DNA strand is separated.
Stage two:
35 stages of the following 3 steps;
1) 95 degrees Celsius for 30 seconds. Double strands of DNA are separated.
2) 57 degrees Celsius for 30 seconds. Primers attach at ends of target DNA segment.
3) 72 degrees Celsius for 30 seconds. DNA polymerase activates and replicates target segment of DNA.
Stage three:
72 degrees Celsius for 3 minutes. Replicate strands attach to each other.
Final Hold:
4 degrees Celsius. PCR reaction is stopped.


DNA Sample Set-up
Two samples of DNA from two separate patients are tested. Patient ID 77842 is the first and patient ID 26204 is the second. A control for both the positive and negative patient is also used.

Positive control:
cancer DNA template
Tube label: P
Patient 1
ID: 77842
Tube label: A1
Patient 1
ID: 77842
Tube label: A2
Patient 1
ID: 77842
Tube label: A3
Negative Control:
no DNA template
Tube label: N
Patient 2
ID: 26204
Tube label: B1
Patient 2
ID: 26204
Tube label: B2
Patient 2
ID: 26204
Tube label: B3


DNA Sample Set-up Procedure
Step 1: Gather all materials
Step 2: Label
Step 3: Pipette
Step 4: Put into PCR
Step 5: Connect to computer
Step 6: Run program
Step 7: Wait for program to finish and clean up

PCR Reaction Mix

  • Taq DNA polymerase
  • MgCl2
  • dNTP's

DNA Sample/Primer Mix

  • Extracted sample of each patient's DNA
  • All tubes have the same forward primer and reverse primer





Research and Development

PCR - The Underlying Technology


Components of PCR

The polymerase chain reaction is comprised of mainly of a template DNA, primers, TAQ polymerase, and Deoxyribonucleotides. More specifically varying PCR's can contain different mixes depending upon the desired outcome and overall proccesses involved. In more detail the template DNA is the genetic coding which will be targeted for replication by everything else in the mix. In addition the primers are short strands of DNA designed to attach to specific points on the DNA and are highly customizable, this done in a lab. The taq polymerase is an enzyme responsible for binding the dNTP to the DNA strands targeted by the primers. the Deoxyribonucleotides or dNTP's is a form of nucleotides with a deoxribose element which bind to the DNA to form a new strand.

PCR Steps/Process

To begin a PCR, a mix of the components listed above, and also whatever else the specific mix may contain depending is placed with a thermocylcer. After the lid is closed typically the setting are adjusted using a software on a computer. The first step for the PCR is for the mix to be heated to 95°C for 3 minutes. This step allows for the DNA strand to split into two seperate single strands. After this is a step referred to as Denature it is accomplished at 95°C for 30 seconds, this is the step that is repeated on successive cycles, as only the initial step needs to be heated for 3 minutes at 95°C. After Denature, is Anneal where the mix is cooled to around 57°C for 30 seconds, this process allows the primers to attach to the single stranded DNA found in the solution. Following this is the Extend step at 72°C for 30 seconds the DNA polymerase adds the chain of nucleotides or dNTP's to copy the strands. After this step the cycle begins annew on Denature then continues in this manner until the final cycle. Where in the last step the DNA is left at 72°C for 3 minutes letteting the Strands that have been replicated combine and attach to each other. Then finally it is placed in hold at 4°C stopping the reaction.

DNA and Importance

The focus of this reaction is finding a combination of the nucleotides A, T, C, & G. And then replicating them exponentially so that when trying to detect the presence of this strand of genetic code it is much simpler since the shortened strand will greatly outnumber the quantity of full DNA strands within a fixed solution of DNA. Finding the right code relies on primers that have been specalized with the correct matching pairs of nucleotides. I.E. A/T and C/G. It is incredibly important that that these primers are accurate otherwise it would be a wasteful effort to replicate the incorrect strand of DNA and the results will not be usable.

Illustrated with ScreenShots Image:PCRsteps2.png Beginning at top left the polymerase chain reaction is demonstrated. In the first image it shows the DNA as it begins in a double strand. Once it reaches the second image to the left it has been heated over 90 degrees Celsius. This causes the DNA to split into two single strands. After this the PCR/DNA mix is cooled to about 55 degrees and the primers get in between the strands and attached to predetermined sections of the DNA. The solution is then heated to 72 degrees about where the Taq polymerase finds the correct ends of the primers. After the polymerase has attached it then uses the dNTPs floating in the solution to replicate all the subsequent DNA beyond those primers until it reaches the end of strand. Once this is finished to cycle. Begins again by heating to 95 degrees to split the two double strands of DNA into 4 single strands. The reaction continues in the same manner as seen on the second to last image the solution is again cooled allowing for the primers to attach to the section of predetermined code. The reaction is completed in the same manner. By the completion of the 3rd cycle there is specific sequences which are shorter in length than the full DNA strands and longer strands. These sequences will grow exponentially as the cycles progress, resulting in an abundance of the target section of code allowing for the testing of the gene anomaly or sequence in question.

Sources: (http://openpcr.org/what-is-pcr)


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