BME103:T130 Group 5: Difference between revisions
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| [[Image: | | [[Image:BME103_Student_Wade.jpg|100px|thumb|Name: Wade Patrick<br>Machine Engineer]] | ||
| [[Image:BME103student.jpg|100px|thumb|Name: Liann Klein<br>Machine Engineer]] | | [[Image:BME103student.jpg|100px|thumb|Name: Liann Klein<br>Machine Engineer]] | ||
| [[Image:Homecoming_picture_2012.jpg|100px|thumb|Name: Haylee Poncy<br>Protocol Planner]] | | [[Image:Homecoming_picture_2012.jpg|100px|thumb|Name: Haylee Poncy<br>Protocol Planner]] | ||
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'''The Original Design'''<br> | '''The Original Design'''<br> | ||
[[Image:BME103_Group5_Assembly.png|250px|PCR Machine]]<br>( | [[Image:BME103_Group5_Assembly.png|250px|PCR Machine]]<br>The Polymerase Chain Reaction (PCR) machine, shown above, is used to replicate a large quantity of a specific strand of DNA. The PCR Machine performs this task by first splitting up the DNA. Since DNA is double stranded, and melts to become two seperate strands at a certain temperature, the PCR Machine heats the DNA to the specific temperature so that the DNA becomes to seperate strands of DNA. Then the PCR Machine uses primers, which are strands of DNA that contain a certain number of nucleotides, to adhere to the two seperate strips of DNA. Then the polymerase, which is an enzyme used to fill in the holes of DNA, completes the strand, resulting in two seperate, double strands of DNA. <br> | ||
'''Experimenting With the Connections'''<br> | '''Experimenting With the Connections'''<br> | ||
When | When the wire from the board for the LCD screen was unplugged from the main board, the LCD screen on the machine turned off and went blank. | ||
When the white wire that connects the main board of the machine and the temperature system was unplugged, the temperature reading decreased and was not accurate.<br> | |||
'''Test Run'''<br> | |||
We administered a test run on October 25 and followed the protocol provided. Everything ran smoothly as the numbers on the Open PCR display screen matched the numbers shown on the computer. | |||
One minor inconsistency was that the estimated time to complete the test did not match the actual time it took to complete it.<br> | |||
'''Test Run''' | |||
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|}<br><br> | |}<br><br> | ||
'''Flourimeter Measurements'''<br> | '''Flourimeter Measurements'''<br><br> | ||
[[Image:BME103_Group5_Assembly(1).jpg|300px|Flourimeter Assembly]]<br>After we used a cellphone to take pictures of the samples that had a blue LED light shining through them, we processed these images with ImageJ software to find the amount of green light that was produced (IntDen). The results from this ImageJ analysis of the images can be seen below: | |||
( | <br> | ||
{|border="1" cellpadding="5" | |||
|- | |||
! scope="col" | Sample | |||
! scope="col" | Area | |||
! scope="col" | Mean | |||
! scope="col" | IntDen | |||
! scope="col" | RawIntDen | |||
|- | |||
|Negative control | |||
|16268 | |||
|21.125 | |||
|343656 | |||
|343656 | |||
|- | |||
|N.c. background | |||
|16268 | |||
|.326 | |||
|5297 | |||
|5297 | |||
|- | |||
|Positive control | |||
|16800 | |||
|76.351 | |||
|1282697 | |||
|1282697 | |||
|- | |||
|P.c. background | |||
|16800 | |||
|.282 | |||
|4732 | |||
|4732 | |||
|- | |||
|Patient 1, sample 1 | |||
|25464 | |||
|14.135 | |||
|359937 | |||
|359937 | |||
|- | |||
|Patient 1, sample 1, background | |||
|25464 | |||
|0.063 | |||
|1603 | |||
|1603 | |||
|- | |||
|Patient 1, sample 2 | |||
|26924 | |||
|44.721 | |||
|1204073 | |||
|1204073 | |||
|- | |||
|Patient 1, sample 2, background | |||
|26924 | |||
|0.009 | |||
|232 | |||
|232 | |||
|- | |||
|Patient 1, sample 3 | |||
|16958 | |||
|20.176 | |||
|342142 | |||
|342142 | |||
|- | |||
|Patient 1, sample 3, background | |||
|16958 | |||
|0.088 | |||
|1486 | |||
|1486 | |||
|- | |||
|Patient 2, sample 1 | |||
|15276 | |||
|79.489 | |||
|1214276 | |||
|1214276 | |||
|- | |||
|Patient 2, sample 1, background | |||
|15276 | |||
|0.264 | |||
|4040 | |||
|4040 | |||
|- | |||
|Patient 2, sample 2 | |||
|22628 | |||
|72.753 | |||
|1646248 | |||
|1646248 | |||
|- | |||
|Patient 2, sample 2, background | |||
|22628 | |||
|0.066 | |||
|1493 | |||
|1493 | |||
|- | |||
|Patient 2, sample 3 | |||
|22804 | |||
|96.671 | |||
|2204484 | |||
|2204484 | |||
|- | |||
|Patient 2, sample 3, background | |||
|22804 | |||
|0.059 | |||
|1355 | |||
|1355 | |||
|- | |||
|Water | |||
|31856 | |||
|11.147 | |||
|355104 | |||
|355104 | |||
|- | |||
|Water Background | |||
|31856 | |||
|0.021 | |||
|672 | |||
|672 | |||
|- | |||
|Calf Thymus | |||
|13580 | |||
|61.625 | |||
|836872 | |||
|836872 | |||
|- | |||
|Calf Thymus Background | |||
|12212 | |||
|0.05 | |||
|611 | |||
|611 | |||
|- | |||
|} | |||
<br><br> | <br><br> | ||
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| | ||
If the r1789961 SNP is not present, the primer will not bind to the DNA template. Because the primers tell Taq polymerase where to replicate, Taq polymerase will not have anywhere to bind. Replication will not occur with the product of a double-stranded DNA, but linearly. The SYBR Green I dye only binds to double-stranded DNA, so it will not show because it is single-stranded. There would not be enough double-stranded DNA to fluoresce if the cancer gene is not present. Therefore, the test will display a negative result. | If the r1789961 SNP is not present, the primer will not bind to the DNA template. Because the primers tell Taq polymerase where to replicate, Taq polymerase will not have anywhere to bind. Replication will not occur with the product of a double-stranded DNA, but linearly. The SYBR Green I dye only binds to double-stranded DNA, so it will not show because it is single-stranded. There would not be enough double-stranded DNA to fluoresce if the cancer gene is not present. Therefore, the test will display a negative result. | ||
<br> <br> | |||
<center> [[Image:Targetsequence112493.png]] <br> <br> | |||
'''Figure 1 shows the sequence of DNA containing the sequence containing the missense that leads to colon cancer.''' <br> <br> | |||
[[Image:Primersattaching112493.png]] <br> <br> | |||
'''Figure 2 shows the DNA primers specific to the sequence attaching.''' <br> <br> | |||
[[Image:Polymeraseattaching112493.png]] <br> <br> | |||
'''Figure 3 shows Taq polymerase recognizing where to attach due to the primers.''' <br> <br> | |||
[[Image:ReplicatedDNA112493.png]] <br> <br> | |||
'''Figure 4 shows a successful double-stranded replication of the original DNA.''' <br> <br> | |||
<br> | [[Image:ReplicatedDesiredSequence112493.png]] <br> <br> | ||
'''Figure 5 shows the desired sequence replicated after several cycles''' <br> <br> | |||
[[Image:AmplifiedDNA112493.png]] <br> <br> | |||
'''Figure 6 shows the amplification of the sequence to a greater magnitude than Figure 1 after more cycles''' <br> <br> | |||
Source of images: http://learn.genetics.utah.edu/content/labs/pcr/ | |||
<br><br> | <br><br> | ||
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==Results== | ==Results== | ||
<!--- Place two small Image J data images here. One showing the result of Water and the other showing the result of Calf Thymus DNA ---> | |||
<!--- 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. ---> | |||
{| {{table}} | |||
|- style="background:#f0f0f0;" | |||
| '''Sample''' || '''Integrated Density''' || '''DNA μg/mL''' || '''Conclusion''' | |||
|- | |||
| PCR: Negative Control || 338359 || 0 || negative | |||
|- | |||
| PCR: Positive Control || 1277965 || 3.848 || positive | |||
|- | |||
| PCR: Patient 1 ID 68754,, rep 1 || 358334 || 0 || negative | |||
|- | |||
| PCR: Patient 1 ID 68754,, rep 2 || 1203841 || 3.539 || positive | |||
|- | |||
| PCR: Patient 1 ID 68754,, rep 3 || 340656 || 0 || negative | |||
|- | |||
| PCR: Patient 2 ID 66913, rep 1 || 1210236 || 3.565 || positive | |||
|- | |||
| PCR: Patient 2 ID 66913, rep 2 || 1644755 || 5.376 || positive | |||
|- | |||
| PCR: Patient 2 ID 66913, rep 3 || 2203129 || 7.703 || positive | |||
|} | |||
Despite Patient 1's second sample being positive for cancer, patient 1 is more than likely without cancer and this repetition being positive is due to an error like contamination. More testing would be necessary to find out. | |||
KEY | |||
* '''Sample''' = <!--- explain what "sample" means ---> The samples were the various distinct sources of DNA measured. | |||
* '''Integrated Density''' = <!--- explain what "integrated density" means and how you did background subtraction to get this value ---> Using the ImageJ software, the image was split into its various color components. This value represents the amount of 'green' light measured from the sample with the blue LED light shining through the sample and subtracted from the background value, which was 'black' in color. | |||
* '''DNA μg/mL''' = <!--- explain how you calculated this ---> The concentration of DNA in the sample as micro-grams per milliliter. | |||
* '''Conclusion''' = <!--- explain what "Positive" and "No signal" means, relative to the control samples ---> Samples with a concentration above a certain threshold were deemed "positive" for the cancer, while the samples with DNA concentrations below the threshold were considered to be "negative". | |||
Latest revision as of 14:52, 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-UPInitial Machine TestingThe Original Design
When the wire from the board for the LCD screen was unplugged from the main board, the LCD screen on the machine turned off and went blank.
When the white wire that connects the main board of the machine and the temperature system was unplugged, the temperature reading decreased and was not accurate. Test Run
ProtocolsPolymerase Chain Reaction Polymerase Chain Reaction (PCR) is a process that uses DNA polymerase to synthesize a large number of copies of a target DNA sequence. PCR is dependent on short DNA fragments called primers. After the DNA has been denatured by heating and then cooled to a temperature suitable for the primers to bind to their complementary sequences, the primers bind to areas adjacent to each side of the targeted DNA sequence. Once the primers are in place, the polymerase extends them into large complimentary strands. The DNA is then denatured once again, then cooled, the primers bind to the complimentary sequence and then the polymerase extends them. Repeating this process results in an exponential amplification of the target DNA sequence.
Flourimeter Measurements
Research and DevelopmentSpecific Cancer Marker Detection - The Underlying Technology The sequence r17879961 represents a specific sequence where a Thymine is replaced by Cytosine due to a missense mutation on chromosome 22. It affects gene CHK2 that is linked to colorectal cancer. A primer binds to a specific sequence on the template DNA and tells Taq polymerase where to begin reading and adding nucleotides to synthesize a new strand of DNA. Primers are very specific in that they can only bind to a certain sequence. A backwards primer consists of 20 nucleotides that specifically are ACT TCT TAC ATT CGA TAC AT. The forward primer is TGT GAT CTT CTA TGT ATG CA. These primers will only bind to that specific sequence of r17879961 where the Cytosine is present and not the Thymine. If the sequence is present, the primers will bind to both leading and lagging strands of the template DNA. Taq polymerase can then bind and begin synthesizing the strand. The test will come out positive because the DNA will synthesize to create double stranded DNA that the SYBR Green I dye will then bind to. This will cause the DNA to fluoresce and yield a positive result.
If the r1789961 SNP is not present, the primer will not bind to the DNA template. Because the primers tell Taq polymerase where to replicate, Taq polymerase will not have anywhere to bind. Replication will not occur with the product of a double-stranded DNA, but linearly. The SYBR Green I dye only binds to double-stranded DNA, so it will not show because it is single-stranded. There would not be enough double-stranded DNA to fluoresce if the cancer gene is not present. Therefore, the test will display a negative result.
Figure 1 shows the sequence of DNA containing the sequence containing the missense that leads to colon cancer. Source of images: http://learn.genetics.utah.edu/content/labs/pcr/
Results
Despite Patient 1's second sample being positive for cancer, patient 1 is more than likely without cancer and this repetition being positive is due to an error like contamination. More testing would be necessary to find out. KEY
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