BME103:T130 Group 7
(→Initial Machine Testing)
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'''The Original Design'''<br>
'''The Original Design'''<br>
This is a PCR machine (model: Open PCR 1 Jankowski and Perfetto). This machine is capable of replicating DNA through a series of temperature changes over timed cycles.<br>
This is a PCR machine (model: Open PCR 1 Jankowski and Perfetto). This machine is capable of replicating DNA through a series of temperature changes over timed cycles. <br>
Revision as of 19:10, 12 November 2012
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LAB 1 WRITE-UP
Initial Machine Testing
The Original Design
When the PCB board was unplugged from the Open PCR circuit board, the machine's LED display ceased to function.
When the white wire that connects the Open PCR circuit board to 16 tube PCR block was unplugged, the machine's LED display read -40 Celsius indicating that the temperature could not be read.
October 18,2012; The test program labeled "Simple Test", was performed to ensure proper function of the Open PCR machine. It ran through two cycles at a full range of temperatures, no DNA was present for the test run. The test performed without any flaws.
Polymerase Chain Reaction
To amplify samples of DNA, the OpenPCR machine was used to perform a Polymerase Chain Reaction (PCR). This technique works by cycling a mixture of DNA Template, Primers, Taq Polymerase, Magnesium Chloride, and dNTP's through three specific temperatures to create more copies of the desired sequence. After assembling the PCR mixture, the PCR machine was programmed to perform three stages. In the first stage, the samples went through one cycle at 95⁰C for 3 minutes. The purpose of this stage is to warm up the machine and prepare the samples for denaturing. The second stage puts the samples through 35 cycles of 95⁰C for 30 Seconds, 57⁰C for 30 seconds, and 72⁰C for 30 seconds. The purpose of the first part of the second stage is to break apart the hydrogen bonds between the base pairs, denaturing the DNA sequence into two separate strands. The purpose of the low temperature is to allow primers to bind. The purpose of the middle temperature is to create an environment for Taq Polymerase to assemble a new strand. Stage three puts the samples through one cycle of 72⁰C for 3 minutes. There is a final hold of 4⁰C. Take the samples out of the PCR machine. The target sequence has been amplified a million times! To analyze the sequence, see the Fluorimeter section.
To analyze the DNA samples after they've been amplified,
A smartphone with adjustable settings was used for this procedure.
- Set exposure to highest setting
The Fluorimeter aparatus consists of a hydrophobic Teflon surface on a glass slide. There is an array of 3x10 glass circles on the surface to anchor the drops. First, place two drops of the cybergreen solution on row 2, column 1&2 (Figure 1). With a sterile pipet, place two drops of amplified sample DNA on top of the cybergreen solution. Do not cross contaminate DNA samples! Take a picture using the settings listed above. Prepare for another sample by sucking the liquid off the slide using a pipet and moving the slide back by two columns. Continue down the columns with the cybergreen drops, another DNA sample(with a new, sterile pipet), pictures, and preparations for another sample. This procedure will require using two slides.
If the dot has DNA, the dye with LED will make the drop appear green. Water will glow blue, and a negative result will also not be green
Research and Development
Specific Cancer Marker Detection - The Underlying Technology
Polymerase Chain Reaction, or PCR, is used to ampllify a specific segment of DNA, in this case the segment known to code for cancer. A primer, which is an artifically synthesized piece of complementary DNA, binds to the desired DNA segment, and the enzyme taq polymerase catalyzes the replication of the DNA strand using dNTPs (free bases). This is repeated over and over to produce many copies of the DNA. Once the copies are made, a fluorescent dye that only bonds to DNA double strands is added to the solution. If a solution that shows fluorescence by using a fluorimeter, then that DNA sample contains the cancer-associated sequence.
The single nucleotide polymorphism, or SNP, that is linked to cancer is rs17879961. The DNA base sequence associated with cancer is ACT, while the non-cancer sequence is ATT. PCR works to identify ACT from ATT because the primers, which are complementary to DNA strands containing cancer-positive ACT, will not bind to DNA containing ATT, and instead of DNA double strands, the solution will only contain single strands. The fluorescent dye will only bind to double strands and will identify those.
These Images were taken using a HTC evo4G using the settings described under flourimeter measurements.
Water and SYBR green
SYBR green and Calf Thymus