The Original Design )
Original design of the Open PCR machine showing inner mechanisms. As the image shows, this Open PCR machine primarily consists of 5 parts: the LCD screen, heated lid, heater, circuit board, and fan. While the machine is portable and easy to use, the design is fragile and has a high failure rate, along with several other design flaws.
Experimenting With the Connections
When we unplugged the LCD screen from the circuit board, the machine's screen stopped displaying.
When we unplugged the white wire that connects the circuit board to the heated lid, the machine stopped controlling the temperature.
Test Run
Our group had machine #1. During our first test run on October 24, 2012, the machine's fan would not work and therefore we could not complete the DNA replication.
Protocols
Polymerase Chain Reaction
How PCR Works
Polymerase chain reaction (PCR) is a process that amplifies minute quantities of DNA in order to obtain a sufficient number of samples for analysis. DNA is a useful health marker and can predict the likelihood that a patient has cancer. During PCR, the double helix structure is unzipped to expose the bases. DNA primer is added to the DNA solution and binds to the gene that causes cancer. Because a non-cancer gene has a different nucleotide sequence from the cancer gene, the primer will not be able to attach to the exposed bases, so the DNA cannot be amplified. DNA amplification involves a sequence of steps called thermal cycling.
Thermal Cycling
1. To separate complementary base pairs, the sample was heated at 95°C for two minutes.
2. During annealing, the temperature was decreased to 57°C to allow the specific primers to attach. This step usually lasts between 30 seconds and one minute.
3. During extension, the temperature is increased to 72°C for one minute to allow Taq DNA polymerase to bind deoxynucleoside triphosphates (dNTPs) on the template DNA, lengthening the synthetic strand.
4. To obtain a sufficient number of samples, the process was repeated 30 times.
Patient 1
Replicate 1
ID: 92336
Gender: Male
Age: 58
Patient 1
Replicate 2
ID: 92336
Gender: Male
Age: 58
Patient 1
Replicate 3
ID: 92336
Gender: Male
Age: 58
Patient 2
Replicate 1
ID: 44606
Gender: Male
Age: 47
Patient 2
Replicate 2
ID: 44606
Gender: Male
Age: 47
Patient 2
Replicate 3
ID: 44606
Gender: Male
Age: 47
Flourimeter Measurements
Research and Development
Specific Cancer Marker Detection - The Underlying Technology
The primer sequence of the single nucleotide polymorphism (SNP) that is linked to colorectal cancer is GGAAGTGGGTCCTAAAAACTCTTACA[C/T]TGCATACATAGAAGATCAGAGTGGC. The gene being affected is CHK2 (checkpoint kinase 2). The allele change is from T to C, which signifies the cancer sequence. The cancer sequence-binding primer, or the reverse primer, is AACTCTACA[C]TGCATACAT. The coordinate of the cancer base pair "C" is at 29,121,087 of the DNA sequence. 20 base pairs (bp) to the left of the cancer sequence was TA, which occurred at coordinate 29,121,067.
Baye's reasoning and statistical formulas can be applied to find the link between the development of cancer and the presence of the cancer gene. In a sample size of 180 patients, 1.1% of contained a single copy of the colorectal cancer (CRC) gene in their DNA (C/T) and 98.9% had no copy of the cancer gene (T/T). According to Baye's rule, the probability of having cancer and also expressing the "C" cancer gene is 1.1% when the probability of expressing the "C" gene and also having cancer is 7.8%, the probability of having cancer is unknown, and standard probability of having cancer over the population is 5.3%. Therefore, the probability of having cancer with the "C" gene is 0.74%.
(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.)