BME103:T130 Group 13: Difference between revisions
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'''The Original Design'''<br> | '''The Original Design'''<br> | ||
[[Image: | [[Image:Group_13_Thursday_130_PCR_labels.png|500px|]]<br> | ||
PCR Machines allow us to make copies of different DNA samples by heating the DNA to the point of denaturing. Once the two strands of DNA are separated, an enzyme builds complimentary strands using the original strands as a template. The PCR Machine is able to be programmed to running multiple cycles of this heating phase. Each cycle results in doubling the previous amount, so by the end of the 30th cycle, you have just over 1,000,000,000 (one billion) copies of the original DNA. This allows the DNA to be analyzed and tested for any defects. | PCR Machines allow us to make copies of different DNA samples by heating the DNA to the point of denaturing. Once the two strands of DNA are separated, an enzyme builds complimentary strands using the original strands as a template. The PCR Machine is able to be programmed to running multiple cycles of this heating phase. Each cycle results in doubling the previous amount, so by the end of the 30th cycle, you have just over 1,000,000,000 (one billion) copies of the original DNA. This allows the DNA to be analyzed and tested for any defects. | ||
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<br>6) Go to your desktop and double-click on the '''ImageJ icon''' and when ImageJ opens, go to the top left of the bar and click on '''File''', next click '''Open'''. | <br>6) Go to your desktop and double-click on the '''ImageJ icon''' and when ImageJ opens, go to the top left of the bar and click on '''File''', next click '''Open'''. | ||
<br>7) A folder will appear on your screen, and on the left click the '''Libraries''' icon, next double-click the '''Pictures''' icon. In the Pictures folder, find the ImageJ Pictures folder you previously created and double-click on that folder. | <br>7) A folder will appear on your screen, and on the left click the '''Libraries''' icon, next double-click the '''Pictures''' icon. In the Pictures folder, find the ImageJ Pictures folder you previously created and double-click on that folder. | ||
<br>8) In the ImageJ Picture folder, '''select an image''' (you can only select one image at a time) and click '''Open'''. In a few seconds, the image will | <br>8) In the ImageJ Picture folder, '''select an image''' (you can only select one image at a time) and click '''Open'''. In a few seconds, the image will appear on your screen. | ||
<br><br>[[Image:BME103_Group13_Assembly.jpg| | <br><br>[[Image:BME103_Group13_Assembly.jpg|400px|Fluorimeter Assembly]] | ||
==Research and Development== | ==Research and Development== | ||
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<!--- Place two small Image J data images here. One showing the result of Water and the other showing the result of Calf Thymus DNA ---> | <!--- Place two small Image J data images here. One showing the result of Water and the other showing the result of Calf Thymus DNA ---> | ||
[[Image: | [[Image:Imajejresults.png|500px|thumb|The results of Image J analysis of SYBR Green.]] | ||
<!--- Enter the values from your group's Data Analyzer table below. E6, F6, etc. are the excel cells from which you should copy your data. ---> | <!--- Enter the values from your group's Data Analyzer table below. E6, F6, etc. are the excel cells from which you should copy your data. ---> | ||
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| PCR: Positive Control || 1233507 || 5.884 || Positive | | PCR: Positive Control || 1233507 || 5.884 || Positive | ||
|- | |- | ||
| PCR: Patient 1 ID | | PCR: Patient 1 ID 65685, rep 1 || 504095 || 2.404 || Negative | ||
|- | |- | ||
| PCR: Patient 1 ID | | PCR: Patient 1 ID 65685, rep 2 || 357380 || 1.705 || Negative | ||
|- | |- | ||
| PCR: Patient 1 ID | | PCR: Patient 1 ID 65685, rep 3 || 801881 || 3.825 || Negative | ||
|- | |- | ||
| PCR: Patient 2 ID | | PCR: Patient 2 ID 58278, rep 1 || 375662 || 1.792 || Negative | ||
|- | |- | ||
| PCR: Patient 2 ID | | PCR: Patient 2 ID 58278, rep 2 || 472779 || 2.255 || Negative | ||
|- | |- | ||
| PCR: Patient 2 ID | | PCR: Patient 2 ID 58278, rep 3 || 407989 || 1.946 || Negative | ||
|} | |} | ||
Latest revision as of 13:38, 15 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-UPPCR Machines allow us to make copies of different DNA samples by heating the DNA to the point of denaturing. Once the two strands of DNA are separated, an enzyme builds complimentary strands using the original strands as a template. The PCR Machine is able to be programmed to running multiple cycles of this heating phase. Each cycle results in doubling the previous amount, so by the end of the 30th cycle, you have just over 1,000,000,000 (one billion) copies of the original DNA. This allows the DNA to be analyzed and tested for any defects.
The Open PCR Machine we received was number 13. When we unplugged the PCB Board of the LCD from the Open PCR Circuit Board, the machine's LCD screen turned off. When we unplugged the white wire that connects the Open PCR Circuit Board to the 16 Tube PCR Block, the machine could not register or measure the temperature. The LCD screen displayed seemingly random numbers including -40 degrees Celsius (which is not possible because the Open PCR Machine was not changing the temperature at that time).
On October 18, 2012, our group first tested the Open PCR Machine number 13. At first, the machine seemed overwhelming in its design. However, after following the instructions and advice from peers and professors, we were able to determine how to properly setup, program, and run a simple test.
ProtocolsPolymerase Chain Reaction Polymerase Chain Reaction is a technology that amplifies a single piece of DNA. This technology works very similarly to the natural DNA replication cycle. One PCR cycle consists of three basic steps: denaturation, annealing and extension. In the denaturation step, heat (usually about 95 degrees Celsius) is used to separate the two strands of DNA. Then in the annealing step, the temperature is decreased to 50 degrees Celsius and the DNA primer, specific to the target sequence for that organism, anneal to the separated strand of DNA. The primers mark the beginning and the end of the targeted DNA sequence. Finally, the extension step required the temperature to be raised to 72 degrees Celsius so that the DNA polymerase is activated. The DNA polymerase begins synthesis at the DNA primer. This results in two double stranded target DNA sequences. The PCR cycle is repeated many times to amplify the targeted strand. There are typically many cycles that need to take place in the PCR in order to amplify a patient's DNA.
After the DNA has been through the thermal cycler, mix each new DNA sample with the the PCR master mix (Taq DNA polymerase, dNTP's, MgCl2, forward primer, and reverse primer) into 8 different Eppendorf tubes using separate pipettes to reduce contamination (see Table 1).
Table 1
Table 2
Table 3
After assembling the fluorimeter, you can now determine if you've amplified the targeted DNA in your PCR experiment. Using the Fluorimeter, you can calculate the relative amount of DNA through fluorescence, which is generated by excitation and emission wavelengths. In order to detect fluorescence when dsDNA is present, you'll be using SYBR Green I because it's more safer compared to other dyes. With that being said, gloves must be worn when handling any liquid containing SYBR Green I. The fluorimeter itself is a very simple machine because it uses optical caustic, a special type of optics that completely removes the need for lasers, mirrors, or lenses. Also the flourimeter is battery-powered, lightweight and portable; this allows every student to have one of these at their lab table. Following the steps below, you can easily learn how to dye your amplified DNA.
Research and DevelopmentSpecific Cancer Marker Detection - The Underlying Technology The reason that DNA with the cancer-associated SNP (single nucleotide polymorphism) rs17879961 will produce a DNA signal while DNA without the SNP will not produce a DNA signal lies in the arrangement of nucleotides at the molecular level. More specifically, the lack of a DNA signal is due to the inability of the reverse primer to bind to the forward strand during the annealing phase of PCR. To detect the cancer-associated sequence of r17879961, the reverse primer is used. This is because the cancer-associated mutation is represented by a single nucleotide in a particular triplet: instead of the normal ATT, the middle T mutates into a C, thus rendering a triplet of ACT (which one can see in the reverse primer shown above). In contrast, the normal sequence with the normal triplet ATT would read . At the protein level, this mutation of 1 nucleotide changes the coded protein from isoleucine to threonine. As a result, the primer will not attach to the normal DNA sequence as it will not have the corresponding nucleotides (AGT) on the forward strand in the particular section of DNA that the mutated sequence would have, but will rather have the triplet AAT. When attempting to replicate DNA during PCR, two primers are required in order to facilitate extension of both the forward and reverse DNA strands. As mentioned above, the reverse primer for the cancer associated sequence would be , having the mutated C instead of the T. A forward primer is also required in order to facilitate extension of the reverse strand. It is usually around 200 base pairs from the mutated DNA sequence and is also 20 base pairs long. For this strand of DNA, a suitable forward primer would be: . With these two primers in place, Taq polymerase can properly do its job by attaching free dNTP’s to the forward and reverse DNA strands during the extension phase of PCR. An illustration of this concept is shown below: An important part of any test is its reliability. If a test or diagnostic does not have a level of reliability, it will be ineffective in identifying populations that pass or fail the test. To measure the reliability a diagnostic will have on a certain population, a theorem known as Bayes Rule is utilized. This equation calculates the probability that A will be true given B. For example, A = hc = people who have cancer in a population while B = C = people in the population who have the rs17879961 SNP; therefore Bayes’ Rule would provide the probability that people who have cancer in population will ALSO have the rs17879961 mutation [p(hc|C)]. In this case, the percentage of people who possess the mutation and have cancer is known [p(C|hc)] along with the percentage of people with the mutation in the population of Finland [p(C)]. Therefore, the only percentage that would need to be found would be the percentage of people who have cancer in Finland [p(hc)] for Bayes’ Rule to work. This example of Bayes’ Rule is illustrated below:
Results
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