BME103:T130 Group 13

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'''Flourimeter Measurements'''<br>
'''Flourimeter Measurements'''<br>
<br>DNA Measurement Equipment Tester
<br>Fluorimeter Assembly Procedure
<br>1) Turn on the excitation light using the switch for the blue LED.
<br>1) Turn on the excitation light using the switch for the blue LED.
<br>2) Place a smart phone on the cradle at a right angle from the slide.
<br>2) Place a smart phone on the cradle at a right angle from the slide.

Revision as of 00:42, 2 November 2012

BME 103 Fall 2012 Home
Lab Write-Up 1
Lab Write-Up 2
Lab Write-Up 3
Course Logistics For Instructors
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Name: Sudarshan IyerResearch and Development Specialist
Name: Sudarshan Iyer
Research and Development Specialist
Name: Ujwala VakaExperimental Protocol Planner
Name: Ujwala Vaka
Experimental Protocol Planner
Name: Emily HerringExperimental Protocol Planner
Name: Emily Herring
Experimental Protocol Planner
Name: Garrett ReppOpen PCR Machine Engineer
Name: Garrett Repp
Open PCR Machine Engineer
Name: Joseph Del RosarioOpen PCR Machine Engineer
Name: Joseph Del Rosario
Open PCR Machine Engineer


The Original Design

Experimenting With the Connections

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.

Test Run

On October 18, 2012, our group first tested the Open PCR Machine. 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.


Polymerase 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 DNA into two strands. 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.
Cycle 1: The PCR master mix contains Taq DNA polymerase, dNTP's, MgCl2, forward primer, and reverse primer. The thermal cycler heats up to 95 degrees Celsius, or 203 degrees Fahrenheit, which is almost at boiling point. At this temperature, DNA double helix separates, creating two single-stranded DNA molecules. Gradually the temperature begins to cool to 50 degrees Celsius so the primers will attach. Then the temperature is raised to 72 degrees DNA polymerase is activated and locates primers attached to the single-strand DNA, which will then begin to add complementary nucleotides onto the strand. This process continues until it gets to the end of the strand and falls off.
Cycle 2: The same three steps occurring in cycle happen in cycle 2. The temperature is raised again to separate the DNA strands, the temperature is lowered so that the primers may attach, and the temperature is raised again slightly to stimulate DNA polymerase to copy the strand.
Cycle 3:the two desired fragments begin to appear—two strands that begin with primer one and end with primer two—and these are the DNA copies of the segment of DNA you’ve targeted. These products will increase (become the majority) as the cycle continues.
Cycle 4: At the end of this cycle, you‘ll have 8 fragments that contain only your target sequence.
Cycle 5: At the end of this cycle, you‘ll have 22 fragments that your target sequence and only ten longer length copies.
After 30 cycles there are over a billion fragments that contain only your target sequence and only 60 copies of the longer length molecules. You now have a solution of nearly pure target sequence.

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

We ran a total of 8 samples. We made 3 samples from Patient 1 (ID#65685) was female and aged 58. We made 3 samples from Patient 2 (ID#58278) who was also female and aged 53. The remaining 2 samples were the positive and negative controls. The positive control for this experiment was the cancer DNA template and the negative control was the no DNA template.

Flourimeter Measurements

Fluorimeter Assembly Procedure
1) Turn on the excitation light using the switch for the blue LED.
2) Place a smart phone on the cradle at a right angle from the slide.
3) Adjust your camera settings as follows:turn off the flash, set the ISO to 800 or higher, increase the exposure to the maximum, and turn off autofocus (optional).
4) Move the smartphone in the cradle as close possible to the first two rows of the slide so that you will get a clear image.
5) The pipette should be filled with liquid only to the bottom of the black line. Then use the pipette to place two drops of water (each drop should be between 130-160 microliters) in the middles of the first two rows of the slide.
6) Move the slide so that the blue LED light is focused on the the drops of water to the middle of the black fiber optic fitting on the other side of the drop.
7) Cover the fluorimeter with the light box so that much of the stray light will removed, but make sure you can still access your smartphone to take pictures.
8) While being careful not to move the smartphone, take three picture of the water droplet.
9) When removing the light box, be careful not to move the smartphone because that could make the analysis more complicated.
10) Use a clean plastic pipette to remove the water droplets from the slide.
11) Push the slide in so that you are npo in the next set of two holes.
12) Repeat steps 5-10 four more times in 5 different positions.
13) Record the following: type of smartphone used, the distance from the base of the smartphone cradle to the measurement device (in cm), and attach one image for each position of the drop (5 images total).

Research and Development

Specific Cancer Marker Detection - The Underlying Technology

The reason that the cancer-associated sequence of r17879961 will produce a DNA signal while the non-cancer DNA sequence of the same SNP (single nucleotide polymorphism) will not produce a DNA signal lies in the arrangement of nucleotides at the molecular level. 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 AAC TCT TAC ACT CGA TAC AT 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 you can see in the reverse primer shown above). 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 r17879961 DNA sequence as it will not have the corresponding base pairs (TGA) in the particular section of DNA that the mutated sequence would have.

(BONUS points: Use a program like Powerpoint, Word, Illustrator, Microsoft Paint, etc. to illustrate how primers bind to the cancer DNA template, and how Taq polymerases amplify the DNA. Screen-captures from the OpenPCR tutorial might be useful. Be sure to credit the source if you borrow images.)


(Your group will add the results of your Fluorimeter measurements from Week 4 here)


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