BME103:T930 Group 11
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! Total Volume || 100.0 μL
! Total Volume || 100.0 μL
Revision as of 01:19, 15 November 2012
|BME 103 Fall 2012|| Home |
Lab Write-Up 1
Lab Write-Up 2
Lab Write-Up 3
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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.
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.
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.
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.
For the PCR process to work, a template DNA which is the sample that you want to test for, is first needed. Primers that could identify the sequence that you want are also needed. Magnesium Chloride is also used to to help Taq polymerase bind the nucleotides (dNTP's) that are needed to replicate the 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.)