BME103:T930 Group 11

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Contents

OUR TEAM

Name: Tony NguyenMachine Tester
Name: Tony Nguyen
Machine Tester
Name: Ryelle PattuinanResearch and Development
Name: Ryelle Pattuinan
Research and Development
Name: Kenze CaulfieldImageJ Technician
Name: Kenze Caulfield
ImageJ Technician
Name: Jackie JanssenRole(s)
Name: Jackie Janssen
Role(s)
Name: Ben HookRole (s)
Name: Ben Hook
Role (s)
Name: Samantha BoccasiniMachine Tester
Name: Samantha Boccasini
Machine Tester

LAB 1 WRITE-UP

Initial Machine Testing

The Original Design

This is an Open PCR machine. The purpose of the machines is to fluctuate the temperature of different samples of DNA strands to separate them into a single strand. It allows for a way to amplify DNA sequences by this separation and heating them with primers and DNA polymerase. When raised to a high enough temperature DNA melts, which is where it separates into the two strands, and primers and DNA polymerase are used to fill in the holes and attach to the now single-stranded DNA. The machine is able to amplify a specific sequence of DNA up to 1 billion times. Because of this ability, scientists and investigators can use it to amplify these sequences even if only a small amount of DNA is provided. Single roots in hair or even microscopic splatters of blood left such as at scenes of crime are actually ample amounts of DNA for PCR.


Experimenting With the Connections

When we unplugged (part 3) from (part 6), the machine's display screen didn't turn on or show up.

When we unplugged the white wire that connects (part 6) to (part 2), the machine's temperature reading was altered compared to the accurate values from the computer.


Test Run


We first tested Open PCR on October 25, 2012. The machine was accurate and precise but the cycling was slow.




Protocols

Polymerase Chain Reaction

The Polymerase Chain Reaction works by attaching MgCl to taq enzyme in the solution and then pulling deoxy nucleotide diphosphates, binding them to the strand that is already present. This gives a base to the DNA that is already there.

DNA amplification contains three important steps: heat denauturation,primer annealing, and primer extension.
Step One: Heat Denauturation-Heat is applied to the DNA and the two strands separate.
Step Two:Primer Annealing-With excess dNTPs, oligonucleotides are added.
Step three: Primer Extension-DNA polymerase is added and new strands of DNA are synthesized. The DNA strands combine with the nucleotides to form completed, complimentary strands of DNA.
PCR master mix contains Magnesium Chloride, all four Nucleotides needed to create DNA, and reaction buffers.

Chart One: Reagents and Their Volumes
Reagent: volume
Template DNA(20 ng): .2uL
10 uM forward primer: 1.0 uL
10 uM reverse primer: 1.0 uL
GoTaq master mix: 50.0 uL
DH2O: 47.8 uL
Total Volume: 100.0 uL

Patient one's ID number is 12123, male, and 61 years old. Patient two's ID is 21312, female, and 56 years old.


Flourimeter Measurements
The Flourimeter was assembled by taking the black box, flipping it upside down so that the rest of the materials can be placed inside the box with the flap blocking out excess light. The glass slide was then placed into the flourimeter so that the light of the flourimeter was on the first two rows of holes in the glass slide. The flourimeter was then placed inside the overturned black box, and the phone was placed on the stand. The camera on the phone is lined up with the middle row of holes in the glass slide. After the pictures of the droplets of DNA and SYBR green solution were taken, they were sent to a computer through e-mail and downloaded.

Image:flourimeter11.jpg



Research and Development

Specific Cancer Marker Detection - The Underlying Technology

Polymerase Chain Reaction or PCR for short is a method used to view a short segment of DNA and then amplify it by generating thousands of copies of the specific sequence being focused on. This method operates by fluctuating the temperature in order to allow for certain processes to occur within the DNA.

95 degrees Celsius - the initial temperature used to separate the strands of the DNA.
57 degrees Celsius - the temperature is dropped to this point in order to allow the primers to bind to the individual strands of DNA.
72 degrees Celsius - the temperature is again increased to this point to allow the Taq polymerase to bind and replicate the strands of DNA.

PCR can be used for a variety of purposes, in this case however, PCR was used in order to determine whether or not a specific gene codes for cancer or not. If a cancer gene is located, then it will yield a positive result because the primers only bind to a certain sequence in the DNA and if that certain sequence codes for cancer, then it will produce a positive result indicating that it was located in that sequence.

The specific sequence that was focused on was r17879961 with the sequence AACTCTTACA[C]TGCATACAT instead of being AACTCTTACA[T]TGCATACAT. The change is from ACT to ATT. The change from the C base into the C base is a missense where the normal T mutates into the cancer C.

To identify whether such mutation occurred in the sequence using PCR, primers should first be designed to bind into the specific sequence. The primer needed for the sequence. The forward primer needed for the sequence is TGGTATAAGACATTCCTGT while the reverse primer should be AACTCTTACACTGCATACAT.

Using PCR, the primer will attach to this specific sequence, checkpoint kinase 2 in the 22nd chromosome. Once the thermocycling ends, this sequence will be amplified for each samples. The fluorescent green dye is used to tell whether or not the result is positive or negative. Like explained earlier, if the sample carries the mutation, then the sample would test positive. If it does not, then the sample would test negative because the primers would not be able to bind to the DNA because it does not contain the proper sequence.

This specific sequence is reported to be susceptible too breast and colorectal cancer. It is also linked to Li-Fraumeni syndrome, Osteosarcoma and even prostate cancer. It was also reported that the non-mutated form of the gene is found in about 98.9% of the population while the mutated form is found in 1.1% of the population. Furthermore, the mutated gene is found in 7.8% of patients with colorectal cancer and 5.3% of the rest of the population without colorectal cancer.

(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.)




Results

Sample Integrated Density DNA μg/mL Conclusion
PCR: Negative Control E6 F6 G6
PCR: Positive Control E7 F7 G7
PCR: Patient 1 ID 12123, rep 1 E8 F8 G8
PCR: Patient 1 ID 12123, rep 2 E9 F9 G9
PCR: Patient 1 ID 12123, rep 3 E10 F10 G10
PCR: Patient 2 ID 21312, rep 1 E11 F11 G11
PCR: Patient 2 ID 21312, rep 2 E12 F12 G12
PCR: Patient 2 ID 21312, rep 3 E13 F13 G13


KEY

  • Sample =
  • Integrated Density =
  • DNA μg/mL =
  • Conclusion =
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