BME103:T930 Group 6

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'''Flourimeter Measurements'''<br>
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'''Fluorometer Measurements'''<br>
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[[Image:Biosetup.jpg|250px|thumb|Assembled Flourimeter]]
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[[Image:Biosetup.jpg|250px|thumb|Assembled Fluorometer]]
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''Flourimeter Assembly Procedure''
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''Fluorometer Assembly Procedure''
1. Remove lid from black box and invert the box, open the front flap.
1. Remove lid from black box and invert the box, open the front flap.

Current revision

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

Contents

OUR TEAM

Name: Nicholas Sterkowitz
Open PCR machine engineer
Name: Dominic Ilardi
Open PCR machine engineer
Name: Alexandra Nazareno
Experimental Protocol Planner
Name: Amanda Sweig
Experimental Protocol Planner
Name: Taylor Deegan
Research and Development(s)

LAB 1 WRITE-UP

Initial Machine Testing

The Original Design
The OpenPCR is an enclosed DNA processing and replication tool. It uses cycles of heating and cooling to replicate the desired DNA. The heating unit, LED controls, and all processing components are encased in a wooden box. A hinged lid covers and insulates the DNA heating plate to facilitate the replication process.

Open PCR Machine

Experimenting With the Connections

When we unplugged the LED interface cable from the processing chip, the LED screen went blank.

When we unplugged the white wire that connects the processing chip to the heating block, the displayed temperature was no longer correct.

Test Run

We first tested the OpenPCR around 25 October 2012. The first experience was a simple graphic user interface and extremely easy physical set-up. The only issue was that the directions in the manual were not as clear as they could have been, and the machine ran very slow. Despite the speed, this is an effective machine to amplify and replicate DNA.




Protocols

Polymerase Chain Reaction

Polymerase Chain Reaction (PCR) is a biochemical process the replicates and amplifies a desired sequence of DNA. The original DNA strand is pulled apart and primer marks the location of the targeted DNA sequence for polymerase to begin replication of the complementary strands. This process is continued until there are an exponentially increasing amount of the desired strand, allowing it to be closely examined.The PCR reaction relies on thermal cycling, in which repeated cycles of cooling and heating the samples enable the enzymes to appropriately replicate the targeted strand, and carry out the processes previously described.

The Steps:

1. Obtain DNA sample and label them clearly.

2. Using a micro-pipette, transfer the proper amounts of forward primers, reverse primers, dNtP's and Taq Polymerase master mix to each the 8 labeled DNA sample tubes. Be sure to replace the pipette tip after every transfer to avoid contamination.

3. Place the DNA sample tubes into the Open PCR machine.

4. Close lid and tighten screw until it touches the tops of the tubes.

5. Program the machine to run the following cycles:

- Stage 1: 1 cycle, 95 degrees Celsius for 3 minutes

- Stage 2: 30 cycles, 95 degrees for 30 seconds, 57 degrees for 30 seconds, 72 degrees for 30 second

- Stage 3: 72 degrees for 3 minutes

- Final Hold: 4 degrees


PCR Master Mix Components

GoTaq® DNA Polymerase is supplied in 2X Colorless GoTaq® Reaction Buffer (pH 8.5), 400μM dATP, 400μM dGTP, 400μM dCTP, 400μM dTTP and 3mM MgCl2.


Reagent Volume
Temple DNA (20 ng) 0.2 μL
10 μM forward primer 1.0 μL
10 μM reverse primer 1.0 μL
GoTaq master mix 50.0 μL
dH2O 47.8 μL
Total Volume 100.0 μL

Patient Information

The eight samples that were tested consisted of a positive and negative control. The other six had three DNA samples from one patient and three DNA samples from the second patient. The first patient has an ID of 51919, and is a female that is fifty-four years of age. The second patient has an ID of 60627, and is also a female, but with an age of sixty-three.


Fluorometer Measurements

Assembled Fluorometer

Fluorometer Assembly Procedure

1. Remove lid from black box and invert the box, open the front flap.

2. Place glass slide into holder.

3. Using pipette, squeeze two drops of SYBR green dye inside the round glass windows on slide.

4. Add DNA sample to dye.

5. Align the sample so the blue LED shines directly though it focusing the light on the other side.

6. Move holder apparatus inside the box so that no light reaches it.

7. Place smartphone with the flash off onto holder and direct camera lens at the slide apparatus.

8. Take photo with smartphone and process on computer using ImageJ.


ImageJ Procedure

1. E-mail image to computer

2. On the computer open the image file using ImageJ

3. In ImageJ, under the "analyze" tool bar select "Set Measurements"

4. Make sure the following boxes are checked, and no other boxes other than the ones listed: -Area -Mean Grey Value -Integrated Density

5. From the download folder drag the image file into ImageJ

6. When the image is opened in ImageJ from the top drop down menu select "Image" and then "Color" and then "Split Channels"

7. Close out the red and blue channel, only use the green channel

8. With the Oval tool select only the entire drop of liquid, avoiding the background but encompassing every bit of the droplet

9. After the droplet part of the image is selected, go to "Analyze" then "Measure" or press Ctrl + M

10. Now move the circle to the background of the image by clicking and dragging on the circle, be careful not to change its size

11. Finally, save the results as an excel spreadsheet (set by default)




Research and Development

Specific Cancer Marker Detection - The Underlying Technology

Polymerase chain reaction (PCR) is a biochemical technology that is used to amplify a specific piece of DNA by generating thousands to millions of copies of a particular DNA sequence. The method relies on thermal cycling by heating and cooling the DNA so that the double strands break apart and can be replicated.

What are the component of a PCR reaction?

Template DNA: A specific DNA sequence that is trying to be detected.

Primers: A short segment of DNA sequences that is complementary to the target DNA sequence and binds to the targeted nucleotides.

Taq Polymerase: An enzyme that is active at high temperature that begins at the primer and takes complementary nucleotides from the solution and binds them to the "unzipped" strands.

Magnesium Chloride: A cofactor that attaches to the Taq Polymerase to affect the speed of the reaction.

dNTP's: Deoxynucleotide triphosphates; individual nucleotide bases in that solution that are attached to the replicated DNA strands by Taq Polymerase.

What happens during each step of the thermal cycle?

  • At 95° Celsius: The DNA double helix "unzips" to reveal two complementary single strands.
  • At 57° Celsius: Primers attach to the complementary template sequence forming one forward and one reverse primer.
  • At 72° Celsius: Taq Polymerase starts at the primer and replicates a complementary strand of DNA using the individual nucleotide bases in the solution.

The r17879961 cancer-associated sequence that is being tested for is AAACTCTTACACTGCATACA. The bolded C represents the cancer gene that appears because the C took the place of the normal T base that should be present. The one nucleotide base that was changed results in producing the amino acid of threonine instead of isoleucine. Because of the change in the amino acid being produced, a study in Finland has found that this sequence has been link to susceptibility to colorectal cancer. Of the patients tested, 7.8% of the patients with colorectal cancer had the allele while 5.3% of patients without colorectal cancer had the same allele.

Why does a cancer gene produce a positive result while a normal gene produces a negative?

  • Since the primer is purposely selected to bind to the specific sequence of nucleotides that makes up the cancer gene, if the cancer gene is not present in the DNA, then the primers will not attach to the seperated DNA strands and replication will not take place, resulting in a negative result. However, if the cancer gene is present then the primers will attach and Taq Polymerase will begin the replication creating an exponential copies of the desired gene resulting in a positive result.

Relation to Bayes' Rule: Bayers' rule is used to determine the probability of true positives, false positives, and false negatives to predict the reliability of the process in detecting the cancer sequence in patients.<br<

The equation for Bayes' Rule is P(A|B) = P(B|A)P(A) / P(B), wherein P(A) represents the initial belief in the occurrence of A, P(A|B) represents the degree of belief in A with regards to B, an known factor, and P(B|A) / P(B) represents the support B has for A.



Results

Water Test
Calf Thymus DNA Test
Sample Integrated Density DNA μg/mL Conclusion
PCR: Negative Control 922523 15.04219863 Negative for Cancer Gene
PCR: Positive Control 1697280 27.6749978 Positive for Cancer Gene
PCR: Patient 1 ID 51919, rep 1 548666 8.946272278 Negative for Cancer Gene
PCR: Patient 1 ID 51919, rep 2 844532 13.7705164 Negative for Cancer Gene
PCR: Patient 1 ID 51919, rep 3 548257 8.939604429 Negative for Cancer Gene
PCR: Patient 2 ID 60627, rep 1 776092 12.65456799 Negative for Cancer Gene
PCR: Patient 2 ID 60627, rep 2 419660 6.842766065 Negative for Cancer Gene
PCR: Patient 2 ID 60627, rep 3 665364 10.8490926 Negative for Cancer Gene

KEY

  • Sample = A sample is small collection of information regarding the area being researched. For this experiment, the sample was small fragments of DNA from two patients and a positive and negative control to test for a cancer gene.
  • Integrated Density = Integrated density is sum of the values of the pixels in the selected image on ImageJ. In this case, the selected area was the DNA mixture and SYBR Green, and each pixel was given a value. These values of all the pixels in the image were added together to determine the integrated density.
  • DNA μg/mL = The DNA concentration (in micro grams per milliliter) was determined by dividing the integrated density of the given sample by the integrated density of the calf thymus, then multiplying that quotient by two.
  • Conclusion = Based on the DNA concentration, the conclusion indicates whether or not the patient sample contained the cancer gene sequence. Positive means the gene was present, while negative means there is no such gene was detected.




Personal tools
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