BME103 s2013:T900 Group3

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Contents

OUR TEAM

Name: Jonus ReynaResearch and Development
Name: Jonus Reyna
Research and Development
Name: Amnah Alkhan Protocols
Name: Amnah Alkhan
Protocols
Name: Haifa TakrooniMachine Testing
Name: Haifa Takrooni
Machine Testing
Name: studentRole(s)
Name: student
Role(s)
Name: studentRole(s)
Name: student
Role(s)
Name: studentRole(s)
Name: student
Role(s)

LAB 1 WRITE-UP

Initial Machine Testing

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The Original Design
The Open PCR machine that perform Polymerase Chain Reaction is device, that works by heating and cooling the DNA samples multiple times in extreme changes in temperature. This process will allow DNA strands to segregate ,duplicate and recombine to create large quantities of specific primers in DNA sequences that located in the solution . This is image shows the original structural design of the Open PCR(Polymerase Chain Reaction) machine. As this picture shows the Open PCR machine basically consist of 6 important parts which the LCD screen, heating lid, heater, power supply, circuit board, and fan. The PCR machine need to hook up into a computer via a USB wire to allow the user to control the factor of an experiment.

Experimenting With the Connections

We have to make sure that each piece in the PCR machine working correctly. For example, when we unplugged (part 3)which is LCD Screen from circuit board (part 6),the LSD screen won't show any data set.However, when we unplugged the white wire that connects the circuit boar(part 6) to( part 2) which is the heater, the machine won't give proper temperature.

Test Run

On March 5,2013,open PCR machine was first tested by our group. After trying several machines and USB cables we setup our actual working machine which is number 3.Connected and uploading the information from the computer to the Open PCR allow us to run our simple diagnostic test, and observed the good result by the end of the test .




Protocols

Thermal Cycler Program

The PCR tube goes through three different thermal stages:

Stage 1:

The temperature heats up to 95 °C for three minuets

Stage 2:

A) In stage two, the temperature increases to 95 °C, 205 °F. This boiling temperature separates the DNA helix, making two single-stranded DNA molecules.

B) The PCR tube contains many primer sequences beside DNA strands. However, after separating the DNA double helix, the temperature cools down to 57 °C, about 135 °F. At this temperature, the primers immediately look for and lock onto their targets before the stands can reunite. Because when the heat is 57 °C, the stands tries to find each other to pair up.

C) The temperature heats up to 72 °C, around 163 °F. At this temperature the DNA polymerase becomes active. Then starts adding the complementary nucleotides onto each single DNA strands by attaching both the primer and stands. The DNA polymerase keeps doing this step until it gets to the end of the stand and then falls off.

Stage 3:

Temperature is at 72 °C For three minutes.

- These three stages happen in the first cycle, and it keeps repeating it 30 times to get millions copies of DNA stands.


DNA Sample Set-up:

+ +1 +2 +3
- -1 -2 -3

DNA Sample Set-up Procedure

1) 8 tubes contain 50 μL of PCR reaction mix. And another 8 tubes contain DNA Sample/Primer mix.

2) Label the 8 tubes that contain the DNA sample/primer mix (as is shown in the table above).

3) Transfer the whole 50 μL of each PCR reaction mix to the 8-labeled tubes by using transfer pipettes. (used the transfer pipettes once each time.)

4) Now each 8-labeled tube has a mix of PCR reaction and DNA sample/primer. Placed these tubes into PCR machine.

5) Plug in the OCR machine to a computer and set-up the three different stage of the thermal cycler after downloading the "openPCR" application.

6) Click on Run to run the experiment. So the PCR start working.



PCR Reaction Mix

Taq DNA polymerase.

MgCl2.

dNTP’s.

forward primer.

reverse primer.


DNA/ primer mix

Patient’s DNA.





Research and Development

Specific Cancer Marker Detection - The Underlying Technology

Polymerase chain reaction is the process of copying DNA. Scientists may use this process to copy and study known cancerous DNA so that they may find resolutions to gene mutations.


Deoxyribonucleic acid (DNA) is comprised of sugar phosphate chains and four main nucleotides which are guanine, adenine (purines), cytosine and thymine (pyrimidines). A single strand of DNA looks like a twisted rope ladder. The sugar-phosphate chains are like the side ropes of the rope ladder, and the four nucleotides are like the center ropes that extend perpendicularly from one side rope to the other and these “center ropes” are called rungs. Each rung is made-up of two different nucleotides. The nucleotides are base paired so adenine must always be paired with thymine and guanine must always be paired with cytosine. If they are not paired with their respective other, this would be called a mutation and could be cancerous.


The idea is to obtain DNA with a gene mutation from a patient and amplify their DNA so further testing can be done to study, identify or to cure cancer.

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To perform the PCR, DNA is obtained, then a specific sequence of nucleotides is chosen and identified to be copied such as the gene mutation and this will be the template DNA. A forward and reverse primer is ordered to complement the template DNA. Adenine only attaches to thymine and cytosine only attaches to guanine. The DNA is mixed with the primers, dNTPs, Taq DNA polymerase, and magnesium chloride. The mix is heated to 95 °C for 30 seconds which unzips the double helix strand in half between the base paired nucleotides. Then it is cooled to 57 °C for 30 seconds which causes primer anneal binding on the ends of the nucleotides. It is heated to 72 °C for 30 seconds, this causes the Taq polymerase which is catalyzed by the magnesium chloride to attach the buoyant dNTP’s to their complementary base pair. This completes one cycle and produces one copy. Performing this procedure 30 times would give approximately 1 million copies of the template DNA.






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