BME103:W930 Group8: Difference between revisions
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'''Specific Cancer Marker Detection - The Underlying Technology'''<br> | '''Specific Cancer Marker Detection - The Underlying Technology'''<br> | ||
This experiment uses PCR (polymerase chain reaction) to amplify DNA strands to detect certain base pair sequences. First, DNA primers, salts (MgCl2 or Magnesium Chloride, | |||
Our certain sequence [http://www.ncbi.nlm.nih.gov/snp/?term=rs17879961 rs17879961] is related to the susceptibility to [http://omim.org/entry/604373#0002 Breast and Colorectal Cancer]. In the case of PCR amplification and detection, the sequence for rs17879961 would be copied for by a primer. The primer starts the copying going forward and backward, thus the primers would be CCT for the 3' forward and GCC for the 3' backward. This primer would be used to detect the cancer sequence out of the DNA. The patient would have DNA extracted and prepped for PCR. This prep would include the insertion of the primer, dNTPs, taq Polymerase, solution and a salt, such as Mg2+. This solution would be inserted into the PCR machine to be heated/cooled/heated. During this fir heating process the DNA of the patient would be elongated and separated (along with the SNP for the cancer we are looking for). | Our certain sequence [http://www.ncbi.nlm.nih.gov/snp/?term=rs17879961 rs17879961] is related to the susceptibility to [http://omim.org/entry/604373#0002 Breast and Colorectal Cancer]. In the case of PCR amplification and detection, the sequence for rs17879961 would be copied for by a primer. The primer starts the copying going forward and backward, thus the primers would be CCT for the 3' forward and GCC for the 3' backward. This primer would be used to detect the cancer sequence out of the DNA. The patient would have DNA extracted and prepped for PCR. This prep would include the insertion of the primer, dNTPs, taq Polymerase, solution and a salt, such as Mg2+. This solution would be inserted into the PCR machine to be heated/cooled/heated. During this fir heating process the DNA of the patient would be elongated and separated (along with the SNP for the cancer we are looking for). | ||
This experiment heavily employs the use of PCR, or polymerase chain reaction. This process is used to amplify a segment of DNA for in-depth analysis. The process starts by adding a mix of DNA primers and DNA florescent tags that bind to a specific segment of DNA. After these primers bind, DNA polymerase binds to the primer-DNA strand bond and incorporates the florescent tags into the molecule. The polymerase adds base pairs to the base DNA strand and creates an antisense copy of the original strand. Since this process happens for both strands of the DNA, each process essentially duplicates the DNA strand. In order for this reaction to occur, the solution needs to be heated up to allow the double stranded DNA to melt into single strands. Then the solution needs to cool down and let the DNA anneal. Each one of these heating and cooling steps makes up the process of thermal cycling. Each cycle the DNA is replicated once. The sample is run through anywhere between 30 and 50 cycles in order to amplify the DNA and the targeted signal enough to be picked up by an imaging machine. Since the primers only bind to specific segments of DNA, the sample will only light up when the segment of interest is duplicated. In this lab, the SYBER green dye colored the DNA in solution, only fluorescing when bonded to double-stranded DNA. As a result, PCR can be used to identify the presence or absence of a particular DNA sequence. | |||
'''Primer Development:''' | '''Primer Development:''' |
Revision as of 00:44, 14 November 2012
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Group 8 PCR GurusLAB 1 WRITE-UPInitial Machine TestingThe device depicted above is an open PCR(Polymerase Chain Reaction), and is used for the amplification of DNA. It works by heating and cooling the DNA samples allowing for the splitting and recombination of the DNA with the primers located in the solution. To accomplish this, the device connects to the USB port of a computer where the format of the cycles are inputted. From there the device repeatedly heats and cools via the heated lid, heat sink, and fan. The information is then fed to the arduino board which controls the heating and cooling as well as transferring the information to the LCD screen. Experimenting With the Connections When we unplugged part the LCD screen(part 3) from the arduino(part 6), the LCD display went black and ceased to work. When we unplugged the white wire that connects the Ardiono(part 6) to the PCR block (part 2), the machine read a temperature reading of -40˚C, indicating a malfunction and the temperature could not be accurately determined.
ProtocolsPart 1: PCR Protocol
Polymerase Chain Reaction Polymerase Chain Reaction is used to amplify DNA. In order for this process to be successful, Template DNA must be replicated. A template of DNA consists of a strand in which the order of the nucleotides (bases) is known. With this information, it is possible to replicate the DNA strand by using heat, primers, and polymerase. The process begins by combining the DNA and the master mix. The master mix is composed of all of the necessary ingredients for the completion of the PCR process. The DNA is then placed into a PCR machine, or thermal cycler. The DNA is heated in the thermal cycler, allowing it to denature, or separate into two strands. The primers are then added to the template strand. They mark the start and end points of the specific sequance that is being targeted for replication. Next, the cycler is once again heated. This allows for the polymerase enzyme to activate and to begin adding nucleotides to the DNA strand. At the end of this process two strands are created (Essentially each strand of DNA has one side composed of new new nucleotides that are added by the polymerase and the other composed of the original nucleotides, thus allowing for a total product of two strands). The machine is then cooled and re-heated again. This cycle is generally repeated for 20-30 cycles, or until the desired amount of target DNA sequence strands is reached. The composition of the master mix is listed below. Steps to Run PCR
Part 2: Flourimeter Protocol Flourimeter Measurements The flourimeter is used to measure the amount,or intensity of fluorescence. The blue LED light is turned on to begin using the device. A camera phone is placed on to a designated tray. In order for the process to work, the flash settings on the camera phone must be turned off. Also the ISO must be 800 or higher and exposure must be set to its maximum. It helps to turn off auto focus as well. A slide is placed on to the flourimeter. On this slide a drop of water is inserted in the middle of the first two rows using a pipette. Two more drops are added to the first drop. The LED light is aligned with the drop and the flourimeter is then covered using a light box. The light box will remove excess light, allowing for images of the drop of water to be taken. The drop is then removed from the slide.The process of putting drops into the slide is then repeated using the next set of holes on the slide (Move the slide so that the new set of holes is in line with the blue light before putting the next drop onto the slide. For both parts of the process, each drop should be between 130 and 160 micro-liters.) The process of taking photos of the drop is then repeated using the same method previously presented. About three photos should be taken for each drop.
Note: The computer will recognize that it is a picture file and save it as Jpeg, unless otherwise specified. Research and DevelopmentSpecific Cancer Marker Detection - The Underlying Technology This experiment uses PCR (polymerase chain reaction) to amplify DNA strands to detect certain base pair sequences. First, DNA primers, salts (MgCl2 or Magnesium Chloride, Our certain sequence rs17879961 is related to the susceptibility to Breast and Colorectal Cancer. In the case of PCR amplification and detection, the sequence for rs17879961 would be copied for by a primer. The primer starts the copying going forward and backward, thus the primers would be CCT for the 3' forward and GCC for the 3' backward. This primer would be used to detect the cancer sequence out of the DNA. The patient would have DNA extracted and prepped for PCR. This prep would include the insertion of the primer, dNTPs, taq Polymerase, solution and a salt, such as Mg2+. This solution would be inserted into the PCR machine to be heated/cooled/heated. During this fir heating process the DNA of the patient would be elongated and separated (along with the SNP for the cancer we are looking for). This experiment heavily employs the use of PCR, or polymerase chain reaction. This process is used to amplify a segment of DNA for in-depth analysis. The process starts by adding a mix of DNA primers and DNA florescent tags that bind to a specific segment of DNA. After these primers bind, DNA polymerase binds to the primer-DNA strand bond and incorporates the florescent tags into the molecule. The polymerase adds base pairs to the base DNA strand and creates an antisense copy of the original strand. Since this process happens for both strands of the DNA, each process essentially duplicates the DNA strand. In order for this reaction to occur, the solution needs to be heated up to allow the double stranded DNA to melt into single strands. Then the solution needs to cool down and let the DNA anneal. Each one of these heating and cooling steps makes up the process of thermal cycling. Each cycle the DNA is replicated once. The sample is run through anywhere between 30 and 50 cycles in order to amplify the DNA and the targeted signal enough to be picked up by an imaging machine. Since the primers only bind to specific segments of DNA, the sample will only light up when the segment of interest is duplicated. In this lab, the SYBER green dye colored the DNA in solution, only fluorescing when bonded to double-stranded DNA. As a result, PCR can be used to identify the presence or absence of a particular DNA sequence. Primer Development: With the DNA sequence (SNP) below: 5' GGAAGTGGGTCCTAAAAACTCTTACA[C/T]TGCATACATAGAAGATCACAGTGGC 3' The forward primer would be: 3' CCTTCACCCAGGATTTTTGAG 5' The backward primer would be: 3' ATGTATCTTCTAGTGTCACCG 5' This is within the accepted bp primer length (18-22), follows the GC concentration rule (40-60%), follows the GC clamp rule (G or C within 5 bp of 3' to clamp the primer down), and has an annealing temperature of 61 degrees Celsius forward and 59 degrees Celsius backward. These all show that the primers forward and backward for this strand above would work. Bayes Analysis Bayes analysis accounts for probabilities behind a test. In this example, Bayes rule is use to calculate the probability that a test will give a positive result for cancer when cancer is present: [math]\displaystyle{ p(C|T)= p(T|C)*p(C) / p(T|C)*p(C)+p(T|nC)*p(nC) }[/math] This is where: p(C|T)= probability that cancer present when positive test p(T|C)= probability that positive test when cancer present p(C)= probability of cancer p(T|nC)= probability that positive test when cancer not present p(nC)= probability that cancer not present Meijers-Heijboer et al. (2002) showed that having the CHEK2 gene creates a higher risk, but not always cancer. 1.1% have the gene, but do not have the cancer. 5.1% have the gene and have breast cancer. This was done from 718 families that did not have BRCA1 or BRCA2 mutations. This shows that the cancer gene could be present but still would not mean cancer is present. Dong et al. (2003) measured that 28 out of 578 men (4.8%) have a CHEK2 gene mutation and have prostate cancer. Cybulski et al. (2004) found that in Poland, variants of the CHEK2 gene have a strong link to increase risk of prostate cancer. In Poland, the prevalence of the alleles in 4008 cancer cases and 4000 controls shows the importance in the link between the CHEK2 gene variants and prostate cancer. These variables are found from the OMIM entry Checkpoint Kinase 2. Other factors would be inherent in experimental errors. However, the limited amount of data found through PCR done in this experiment does not allow for a calculated percentage.
All images borrowed from Virtual Lab via the University of Utah.
Results
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