BME103:T930 Group 9: Difference between revisions
(32 intermediate revisions by 5 users not shown) | |||
Line 13: | Line 13: | ||
{| style="wikitable" width="700px" | {| style="wikitable" width="700px" | ||
|- valign="top" | |- valign="top" | ||
| [[Image: BME103_Group9_Devraj.jpg |100px|thumb| | | [[Image: BME103_Group9_Devraj.jpg |100px|thumb|Devraj Patel<br> Open PCR Machine Engineer and Research and Development Scientist]] | ||
| [[Image:BME103 Group9 Drew.jpg |100px|thumb| | | [[Image:BME103 Group9 Drew.jpg |100px|thumb|Andrew Hensley <br> Experimental Protocol Planner]] | ||
| [[Image:BME103 Group9 Nathalie.jpg|100px|thumb| | | [[Image:BME103 Group9 Nathalie.jpg|100px|thumb|Nathalie Vitale <br> Experimental Protocol Planner]] | ||
| [[Image:BME103 Group9 Ojeen.jpg|100px|thumb| | | [[Image:BME103 Group9 Ojeen.jpg|100px|thumb|Ojeen Korkes <br> Research and Development Scientist]] | ||
| [[Image:BME103 Group9 Brandon.jpg|100px|thumb| | | [[Image:BME103 Group9 Brandon.jpg|100px|thumb|Brandon Simmons <br> Open PCR Machine Engineer]] | ||
|} | |} | ||
Line 27: | Line 27: | ||
'''The Original Design'''<br> | '''The Original Design'''<br> | ||
[[Image:PCRMachineImage.png]]<br> | [[Image:PCRMachineImage.png]]<br> | ||
This is an image of an '''Open PCR Machine'''. This machine | This is an image of an '''Open PCR Machine''' (Polymerase Chain Reaction). This machine amplicifies the DNA by regulating the temperature of the DNA smaples both heating and cooling samples using its temperature regulating parts such as heat sink, fan and heated lid to the preset temperature and for the time set up beforehand using the set up program. This heating and cooling seperates the DNA strands and allows recombination of the DNA to occur with the primers added in the PCR tubes. | ||
Line 41: | Line 41: | ||
'''Test Run''' | '''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 | 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 accurately controlled and displayed the preprogrammed temperature determined by the software on the computer. The overall successfullness of the machine was good, however it came with one difficulty, fluctuation of time to complete the preprogrammed cycles. | ||
Line 48: | Line 48: | ||
==Protocols== | ==Protocols== | ||
'''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 synthesize a new strand of DNA. This last step is done at 72 degrees Celsius. In the end, the outputs 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] | |||
<br> | <br> | ||
'''Steps to Run PCR'''<br> | '''Steps to Run PCR'''<br> | ||
Line 57: | Line 64: | ||
#Prepare the experiment by inserting the reactants into the PCR tubes. These tubes will consist of the patients DNA, along with the other provided mixing components'''*'''. After filling each tube, put it into the chamber at the top of the machine.<br> | #Prepare the experiment by inserting the reactants into the PCR tubes. These tubes will consist of the patients DNA, along with the other provided mixing components'''*'''. After filling each tube, put it into the chamber at the top of the machine.<br> | ||
#Close and tighten the lid of the chamber.<br> | #Close and tighten the lid of the chamber.<br> | ||
#Customize the settings in the 'Thermal Cycler' program to include three stages: Stage 1 - one cycle that will heat the reactants up to 95 degrees | #Customize the settings in the 'Thermal Cycler' program to include three stages: Stage 1 - one cycle that will heat the reactants up to 95 degrees Celsius for three minutes, Stage 2 - 35 cycles that will heat the reactants to 95 degrees Celsius for 30 seconds, 57 degrees Celsius for 30 seconds, and 72 degrees Celsius for 30 seconds. <br> | ||
#Press start on the | #Press start on the program to begin running the PCR.<br> | ||
#Collect and record data at the completion of the trial.<br> | #Collect and record data at the completion of the trial.<br> | ||
<br> | <br> | ||
'''* Provided Mixing Components'''<br> | '''* Provided Mixing Components'''<br> | ||
a)TaqDNA polymerase (non-recombinant modified form) <br> | a) TaqDNA polymerase (non-recombinant modified form) <br> | ||
b)MgCl2<br> | b) MgCl2<br> | ||
c)dNTP's <br> | c) dNTP's <br> | ||
d)reaction buffers (at optimal concentration for DNA template amplification)<br> | d) reaction buffers (at optimal concentration for DNA template amplification)<br> | ||
<br> | <br> | ||
Line 113: | Line 120: | ||
'''Flourimeter Measurements'''<br><br> | '''Flourimeter Measurements'''<br><br> | ||
[[Image:BME103_Group9_FluorimeterSetup.jpg|400px|Flourimeter Set-up]]<br><br> | |||
'''Flourimeter Assembly Procedure'''<br> | '''Flourimeter Assembly Procedure'''<br> | ||
Line 133: | Line 142: | ||
#Save the images to the computer. <br> | #Save the images to the computer. <br> | ||
#If the computer does not already have Image J installed, the program can be downloaded by going to http://rsb.info.nih.gov/ij/download.html <br> | #If the computer does not already have Image J installed, the program can be downloaded by going to http://rsb.info.nih.gov/ij/download.html <br> | ||
#In Image J, go to file, open, and then select the desired picture. <br> | #In Image J, go to file, open, and then select the desired picture. <br><br> | ||
<br><br> | |||
'''How to Analyze Pictures in Image J'''<br> | |||
#With the desired picture open, go to analyze, set measurements, and select Area Integrated Density and Mean Grey Value.<br> | |||
#Go to image, color, and then split screen.<br> | |||
#In the green channel, use the oval tool to draw an oval around the water drop.<br> | |||
#Go to analyze, measure, and save the results as an Excel file.<br> | |||
#To get background measurements, draw a similar-sized oval somewhere in the background. Then, go to analyze, measure, and save the results as an Excel file.<br><br> | |||
'''ImageJ Software Processing Measurements''' | |||
{| border="1" cellpadding="2" | |||
!width="200"|Sample | |||
!width="200"|Image or Background | |||
!width="200"|Area | |||
!width="200"|INTDEN | |||
!width="200"|RAWINTDEN | |||
|- | |||
|Negative | |||
|Sample 1 | |||
|6724 | |||
|20.639 | |||
|138777 | |||
|- | |||
|Negative | |||
|Background 1 | |||
|6308 | |||
|0.110 | |||
|693 | |||
|- | |||
|Patient 2 | |||
|Sample 2 | |||
|6943 | |||
|31.042 | |||
|215523 | |||
|- | |||
|Patient 2 | |||
|Background 2 | |||
|6432 | |||
|0.206 | |||
|1326 | |||
|- | |||
|Patient 2 | |||
|Sample 3 | |||
|4177 | |||
|43.131 | |||
|180160 | |||
|- | |||
|Patient 2 | |||
|Background 3 | |||
|4177 | |||
|0.009 | |||
|39 | |||
|- | |||
|Patient 2 | |||
|Sample 4 | |||
|8328 | |||
|72.861 | |||
|606790 | |||
|- | |||
|Patient 2 | |||
|Background 4 | |||
|8328 | |||
|0.022 | |||
|180 | |||
|- | |||
|DNA Calf Thymus & Sybrgreen | |||
|Sample 5 | |||
|3745 | |||
|15.377 | |||
|57587 | |||
|- | |||
|DNA Calf Thymus & Sybrgreen | |||
|Background 5 | |||
|3725 | |||
|0.029 | |||
|107 | |||
|- | |||
|Positive | |||
|Sample 6 | |||
|6014 | |||
|74.302 | |||
|446850 | |||
|- | |||
|Positive | |||
|Background 6 | |||
|6014 | |||
|0.017 | |||
|103 | |||
|- | |||
|Patient 1 | |||
|Sample 7 | |||
|4916 | |||
|76.736 | |||
|377325 | |||
|- | |||
|Patient 1 | |||
|Background 7 | |||
|4916 | |||
|0.023 | |||
|112 | |||
|- | |||
|Patient 1 | |||
|Sample 8 | |||
|4292 | |||
|72.757 | |||
|312275 | |||
|- | |||
|Patient 1 | |||
|Background 8 | |||
|4594 | |||
|0.158 | |||
|728 | |||
|- | |||
|Patient 1 | |||
|Sample 9 | |||
|7815 | |||
|35.699 | |||
|278990 | |||
|- | |||
|Patient 1 | |||
|Background 9 | |||
|7815 | |||
|0.015 | |||
|114 | |||
|- | |||
|Water | |||
|Sample 10 | |||
|4844 | |||
|51.982 | |||
|251803 | |||
|- | |||
|Water | |||
|Background 10 | |||
|4784 | |||
|0.010 | |||
|47 | |||
|} | |||
<br> | |||
<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### --> | |||
|} | |||
==Research and Development== | ==Research and Development== | ||
<br> | |||
'''Specific Cancer Marker Detection - The Underlying Technology'''<br> | |||
After studying how the polymerase chain reaction machine works and the results it yields, we have to study how the DNA processed can be used to identify any sort of disease. Specifically, the rs17879961 cancer-associated sequence will produce a DNA signal because of the single nucleotide variation in its gene code. Based on our study of the rs17879961 cancer-associated sequence, we found that the missense mutation in e gene code yields a positive identification marker for cancer when the a single nucleotide C changes to a single nucleotide T. <br><br> | |||
Original Code:<br> | |||
GGAAGTGGGTCCTAAAAACTCTTACA['''''C''''']TGCATACATAGAAGATCACAGTGGC<br><br> | |||
Modified Code: (due to SNP)<br> | |||
GGAAGTGGGTCCTAAAAACTCTTACA['''''T''''']TGCATACATAGAAGATCACAGTGGC<br><br> | |||
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).<br><br> | |||
'' | 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. Bayesian reasoning accounts for the prevalence, sensitivity, specificity and false positive rates of a disease in order to further understand that disease. Sensitivity is the likelihood that a person afflicted with the disease will test positive for it, while specificity is the likelihood that a person without the disease will test negative for it. 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%.<br> | ||
For more infomation, visit http://www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi?rs=17879961.<br><br> | |||
[[Image: BME103_Group9_RNA Primer Binding.jpg |1000px|thumb|center|Summary: This is an image of how RNA primers bind to the cancer DNA template during the replication process. [2]]] Source: [http://matthew2262.files.wordpress.com/2011/04/replication.jpg]<br> | |||
[[Image: BME103_Group9_Taq Polymerase.gif |500px|thumb|center|Summary: This is an image of how Taq polymerases amplify the DNA. [3]]] Source: [http://www2.le.ac.uk/departments/emfpu/genetics/explained/images/PCR-process.gif]<br> | |||
<br>For more information on the PCR DNA replication process, please visit this website: http://learn.genetics.utah.edu/content/labs/pcr/. | <br>For more information on the PCR DNA replication process, please visit this website: http://learn.genetics.utah.edu/content/labs/pcr/. | ||
Line 152: | Line 311: | ||
<br> | <br> | ||
==Results== | |||
== | '''Positive Test''' <br> | ||
[[Image:BME103 Group9 Positivetest.jpg|200px|]] <br><br> | |||
'''Negative Test''' <br> | |||
[[Image:BME103 Group9 Negativetest.jpg|200px|]]<br><br> | |||
'''The Patient Information'''<br> | |||
{|border="1" cellpadding="5" | |||
|- | |||
! scope="col" | Patient Identification Number | |||
! scope="col" | Gender | |||
! scope="col" | Age | |||
|- | |||
|31542 | |||
|Male | |||
|55 | |||
|- | |||
|52125 | |||
|Female | |||
|55 | |||
|} | |||
Line 164: | Line 340: | ||
| '''Sample''' || '''Integrated Density''' || '''DNA μg/mL''' || '''Conclusion''' | | '''Sample''' || '''Integrated Density''' || '''DNA μg/mL''' || '''Conclusion''' | ||
|- | |- | ||
| PCR: Negative Control || | | PCR: Negative Control || 138082.756 || 0.49 || NEG - Water | ||
|- | |- | ||
| PCR: Positive Control || | | PCR: Positive Control || 214199.614 || 1.54 || POS - Calf Thymus | ||
|- | |- | ||
| PCR: Patient 1 ID | | PCR: Patient 1 ID 31542, rep 1 || 377121.108 || 2.70 || POS | ||
|- | |- | ||
| PCR: Patient 1 ID | | PCR: Patient 1 ID 31542, rep 2 || 311547.192 || 2.23 || POS | ||
|- | |- | ||
| PCR: Patient 1 ID | | PCR: Patient 1 ID 31542, rep 3 || 278870.460 || 1.00 || NEG | ||
|- | |- | ||
| PCR: Patient 2 ID | | PCR: Patient 2 ID 52125, rep 1 || 253204.641 || 1.82 || POS | ||
|- | |- | ||
| PCR: Patient 2 ID | | PCR: Patient 2 ID 52125, rep 2 || 180120.594 || 1.29 || NEG | ||
|- | |- | ||
| PCR: Patient 2 ID | | PCR: Patient 2 ID 52125, rep 3 || 606603.192 || 4.35 || POS | ||
|} | |} | ||
KEY | KEY | ||
* '''Sample''' = <!--- explain what "sample" means ---> | * '''Sample''' = <!--- explain what T"sample" means --->The sample is the substance tested using the flourimeter, in the case of this lab the substances used are a positive control, negative control, 3 trials for patient one, and 3 trials for patient 2. | ||
* '''Integrated Density''' = <!--- explain what "integrated density" means and how you did background subtraction to get this value ---> | * '''Integrated Density''' = <!--- explain what "integrated density" means and how you did background subtraction to get this value --->Integrated density is the sum of the pixels in a given are, this is found by finding the product of the area and mean pixel value then subtracting the background. | ||
* '''DNA μg/mL''' = <!--- how you calculated this ---> | * '''DNA μg/mL''' = <!--- explain how you calculated this --->This is the concentration of DNA in the respective sample, this is calculated by multiplying the integrated density by 2 and dividing by the integrated density value of calf thymus. | ||
* '''Conclusion''' = <!--- explain | * '''Conclusion''' = <!--- explain what "Positive" and "No signal" means, relative to the control samples --->A positive signal represents a sample that exhibited the same reaction to sybr green in the lab as our positive control; a negative signal represents a sample that exhibited the same reaction to sybr green as the negative control. | ||
<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### --> | <!-- ##### DO NOT edit below this line unless you know what you are doing. ##### --> | ||
Latest revision as of 12:46, 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 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 accurately controlled and displayed the preprogrammed temperature determined by the software on the computer. The overall successfullness of the machine was good, however it came with one difficulty, fluctuation of time to complete the preprogrammed cycles.
ProtocolsPolymerase Chain Reaction
Source: [1]
Flourimeter Assembly Procedure
How to Open Pictures Using Image J
How to Analyze Pictures in Image J
ImageJ Software Processing Measurements
|
Research and Development
Specific Cancer Marker Detection - The Underlying Technology
After studying how the polymerase chain reaction machine works and the results it yields, we have to study how the DNA processed can be used to identify any sort of disease. Specifically, the rs17879961 cancer-associated sequence will produce a DNA signal because of the single nucleotide variation in its gene code. Based on our study of the rs17879961 cancer-associated sequence, we found that the missense mutation in e gene code yields a positive identification marker for cancer when the a single nucleotide C changes to a single nucleotide T.
Original Code:
GGAAGTGGGTCCTAAAAACTCTTACA[C]TGCATACATAGAAGATCACAGTGGC
Modified Code: (due to SNP)
GGAAGTGGGTCCTAAAAACTCTTACA[T]TGCATACATAGAAGATCACAGTGGC
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. Bayesian reasoning accounts for the prevalence, sensitivity, specificity and false positive rates of a disease in order to further understand that disease. Sensitivity is the likelihood that a person afflicted with the disease will test positive for it, while specificity is the likelihood that a person without the disease will test negative for it. 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.
Source: [2]
Source: [3]
For more information on the PCR DNA replication process, please visit this website: http://learn.genetics.utah.edu/content/labs/pcr/.
Results
Positive Test
Negative Test
The Patient Information
Patient Identification Number | Gender | Age |
---|---|---|
31542 | Male | 55 |
52125 | Female | 55 |
Sample | Integrated Density | DNA μg/mL | Conclusion |
PCR: Negative Control | 138082.756 | 0.49 | NEG - Water |
PCR: Positive Control | 214199.614 | 1.54 | POS - Calf Thymus |
PCR: Patient 1 ID 31542, rep 1 | 377121.108 | 2.70 | POS |
PCR: Patient 1 ID 31542, rep 2 | 311547.192 | 2.23 | POS |
PCR: Patient 1 ID 31542, rep 3 | 278870.460 | 1.00 | NEG |
PCR: Patient 2 ID 52125, rep 1 | 253204.641 | 1.82 | POS |
PCR: Patient 2 ID 52125, rep 2 | 180120.594 | 1.29 | NEG |
PCR: Patient 2 ID 52125, rep 3 | 606603.192 | 4.35 | POS |
KEY
- Sample = The sample is the substance tested using the flourimeter, in the case of this lab the substances used are a positive control, negative control, 3 trials for patient one, and 3 trials for patient 2.
- Integrated Density = Integrated density is the sum of the pixels in a given are, this is found by finding the product of the area and mean pixel value then subtracting the background.
- DNA μg/mL = This is the concentration of DNA in the respective sample, this is calculated by multiplying the integrated density by 2 and dividing by the integrated density value of calf thymus.
- Conclusion = A positive signal represents a sample that exhibited the same reaction to sybr green in the lab as our positive control; a negative signal represents a sample that exhibited the same reaction to sybr green as the negative control.