BME103:T930 Group 9: Difference between revisions
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'''Polymerase Chain Reaction'''<br> | '''Polymerase Chain Reaction'''<br> | ||
PCR, polymerase chain reaction, is a simple tool that one can use to focus in onto a segment of DNA and generate thousands to millions of copies of a particular DNA sequence. In PCR, everything relies heavily on the regulation and variation of temperature. By setting the PCR at specific temperatures, a chain of reactions can take place. Therefore, there are three steps in PCR cycle: denaturation, annealing, and extension. In the beginning, PCR should be heated to 95 degrees Celsius. This allows the double-stranded DNA to be separated and unwinded. Secondly, PCR should be cooled at 57 degrees Celsius because this allows a piece of DNA to bind to DNA product from the initial step. This is done through primer enzymes that allow polymerase to start synthesizing by recognizing and attaching to sequences that are complementary. Lastly, the extension step is where DNA product will continually add bases following the primers until it fully synthesizes a new strand of DNA. This last step is done at 72 degrees Celsius. In the end, the output of these reactions yield up to millions of strands of DNA that can be examined to identify certain types of genes in diseases or utilized for scientific purposes. | PCR, polymerase chain reaction, is a simple tool that one can use to focus in onto a segment of DNA and generate thousands to millions of copies of a particular DNA sequence. In PCR, everything relies heavily on the regulation and variation of temperature. By setting the PCR at specific temperatures, a chain of reactions can take place. Therefore, there are three steps in PCR cycle: denaturation, annealing, and extension. In the beginning, PCR should be heated to 95 degrees Celsius. This allows the double-stranded DNA to be separated and unwinded. Secondly, PCR should be cooled at 57 degrees Celsius because this allows a piece of DNA to bind to DNA product from the initial step. This is done through primer enzymes that allow polymerase to start synthesizing by recognizing and attaching to sequences that are complementary. Lastly, the extension step is where DNA product will continually add bases following the primers until it fully synthesizes a new strand of DNA. This last step is done at 72 degrees Celsius. In the end, the output of these reactions yield up to millions of strands of DNA that can be examined to identify certain types of genes in diseases or utilized for scientific purposes. | ||
Source: [http://openpcr.org/use-it] | |||
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Revision as of 21:42, 12 November 2012
BME 103 Fall 2012 | Home People Lab Write-Up 1 Lab Write-Up 2 Lab Write-Up 3 Course Logistics For Instructors Photos Wiki Editing Help | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
OUR TEAMLAB 1 WRITE-UPInitial Machine TestingThe Original Design
When we unplugged part 3, the LCD, from part 6, the Open PCR circuit Board, the LCD on the machine turned off and no information appeared on the LCD screen.
Test Run On October 25 2012, we conducted our first test on our open PCR machine. We tested the machine to test the operation functionality. The initial test demonstrated the machine heat sink accuratly controlled and displayed the preprogrammed temperaute determined by the software on the computer. The overall successfullness of the machine was good, however it came with one difficulity, fluctuation of time to complete the preporgrammed cycles.
ProtocolsPolymerase Chain Reaction
Source: [1]
Flourimeter Assembly Procedure
How to Open Pictures Using Image J
Research and DevelopmentSpecific Cancer Marker Detection - The Underlying Technology Original Code: Modified Code: (due to SNP) When considering scientific detection of the missense mutation itself, we have found that our DNA sequence rs17879961 is related to the condition of Breast and Colorectal Cancer. Therefore, in the case of PCR detection, the sequence for rs17879961 would be copied for by a primer. The primer starts the copying going forward and backward, with the primer that correlate to the strand of DNA; this primer identify the cancer sequence out of the DNA. Then, the patient would have that strand of DNA extracted and prepared for PCR amplification process. This preparation would include the use of primers, taq Polymerase, solution and dNTPs, and other necessary materials. This solution would be inserted into the PCR machine to be heated/cooled/heated. Eventually, the PCR process would yield multiple strands of the DNA that was initially placed and the SNP part that we had identified. A non-cancer DNA sequence would not produce a signal because the nucleotide variation where a primer would replicate DNA would be out of place; therefore, its process of DNA amplification would occur as normal. Only when we have a mutation, can we identify a signal from the DNA (assuming that we are attempting to detect a normal nucleotide sequence). As mentioned previously, we studied that the cancer marker, rs17879961, in the PCR experiment was correlated to the Breast and Colorectal cancer, but to completely understand the extent of this cancer's SNP to the development of cancer, we need to take a look at the statistics that not only follow Baye's Rule, but also provide useful information about the spread of this type of cancer. Based on conditional probabilities from a population diversity in Finland where the tested sample was 180 people, we found that the frequency of this cancer found in Finland was 7.8%. The genotype of this sequence of C/T in this population was 1.1% and the genotype of T/T was found to be 98.9%. For more infomation, visit http://www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi?rs=17879961. 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
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