BME103:T130 Group 7

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
(Protocols)
(Protocols)
Line 70: Line 70:
The Fluorimeter aparatus consists of a hydrophobic Teflon surface on a glass slide. There is an array of 3x10 glass wells on the surface to anchor drops in position for photographing. First, two drops of the cybergreen solution were placed on row 2, column 1&2 (Figure 1). With a sterile pipet two drops of amplified sample DNA were added to the cybergreen solution. It was important to place the DNA on top of the cybergreen in order to achieve the best results and to avoid contamination of the DNA samples. The picture was then photographed in the dark box with the door shut to eliminate excess background light. The next sample was prepared by sucking the liquid off the slide using a 'waste' pipet. The slide was then moved back by two columns for the next sample. This procedure was replicated for each of the patient samples and two slides were used in their entirety. <br> <br>
The Fluorimeter aparatus consists of a hydrophobic Teflon surface on a glass slide. There is an array of 3x10 glass wells on the surface to anchor drops in position for photographing. First, two drops of the cybergreen solution were placed on row 2, column 1&2 (Figure 1). With a sterile pipet two drops of amplified sample DNA were added to the cybergreen solution. It was important to place the DNA on top of the cybergreen in order to achieve the best results and to avoid contamination of the DNA samples. The picture was then photographed in the dark box with the door shut to eliminate excess background light. The next sample was prepared by sucking the liquid off the slide using a 'waste' pipet. The slide was then moved back by two columns for the next sample. This procedure was replicated for each of the patient samples and two slides were used in their entirety. <br> <br>
Picture and Analysis: <br>
Picture and Analysis: <br>
-
The pictures of each sample were analyzed using the "image j" software to process the images and analyze their content. The pictures, taken with the settings listed above, were upload to the "image j" software. The images were then separated into their composite colors, red, blue, and green. The green portion of the images were then analyzed using the software and its "" was recorded.
+
The pictures of each sample were analyzed using the "image j" software to process the images and analyze their content. The pictures, taken with the settings listed above, were upload to the "image j" software. The images were then separated into their composite colors, red, blue, and green. The green portion of the images were then analyzed using the software and its "INTDEN" was recorded.
Line 76: Line 76:
Interpreting results:
Interpreting results:
-
The cybergreen soluiton allowed the amount of DNA in a sample to be determined by its luminosity. The cybergreen glowed green in the presence of DNA and while this glow was not visable to the naked eye, the smart phone in conjunction with image j was able to quantify the luminosity. The brighter samples contained more DNA and this indicated that the sample reacted with the primers and was cancerous.
+
The cybergreen soluiton allowed the amount of DNA in a sample to be determined by its "INTDEN". The cybergreen glowed green in the presence of DNA and while this glow was not visable to the naked eye, the smart phone in conjunction with "image j" was able to quantify the luminosity in terms of the "INTDEN". The brighter samples contained more DNA and had a higher "INTDEN" value. This indicated that the sample reacted with the primers better and was cancerous.
==Research and Development==
==Research and Development==

Revision as of 21:55, 12 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
Image:BME494_Asu_logo.png

Contents

OUR TEAM

Name: Emily ThompsonResearch & Development Scientist
Name: Emily Thompson
Research & Development Scientist
Name: Vivian BenjesExperimental Protocol Planner
Name: Vivian Benjes
Experimental Protocol Planner
Name: Frances MarrettExperimental Protocol Planner
Name: Frances Marrett
Experimental Protocol Planner
Name: Chris GlassOpen PCR Machine Engineer[1]
Name: Chris Glass
Open PCR Machine Engineer
[1]
Name: Ryan FrantzOpen PCR Machine Engineer[2]
Name: Ryan Frantz
Open PCR Machine Engineer
[2]
Name: Cenric NigburGhost/ TBD
Name: Cenric Nigbur
Ghost/ TBD

LAB 1 WRITE-UP

Initial Machine Testing

The Original Design
This is a PCR (polymerase chain reaction) machine (model: Open PCR 1 Jankowski and Perfetto). This machine is capable of replicating DNA through a series of temperature changes over timed cycles. It uses specialized primers to target specific gene sequences and is capable of amplifying singular stretches of DNA.

Image:PCR Machine.png


Experimenting With the Connections

When the PCB board was unplugged from the Open PCR circuit board, the machine's LED display ceased to function.

When the white wire that connects the Open PCR circuit board to 16 tube PCR block was unplugged, the machine's LED display read -40 Celsius indicating that the temperature could not be read.


Test Run

October 18,2012; The test program labeled "Simple Test", was performed to ensure proper function of the Open PCR machine. It ran through two cycles at a full range of temperatures, no DNA was present for the test run. The test performed without any flaws.




Protocols

Polymerase Chain Reaction

To amplify samples of DNA, the OpenPCR machine was used to perform a Polymerase Chain Reaction (PCR). This technique worked by cycling a mixture of DNA Template, Primers, Taq Polymerase, Magnesium Chloride, and dNTP's through three specific temperatures to create more copies of the desired sequence. After assembling the PCR mixture, the PCR machine was programmed to perform three stages. In the first stage, the samples went through one cycle at 95⁰C for 3 minutes. The purpose of this stage was to initially denature the DNA and allow the primers to act on the DNA. The second stage put the samples through 35 cycles of 95⁰C, 57⁰C, and 72⁰C each for 30 seconds. The purpose of the first part of the second stage is to break apart the hydrogen bonds between the base pairs, denaturing the DNA sequence into two separate strands. The purpose of the low temperature is to allow primers to bind. The purpose of the middle temperature is to create an environment for Taq Polymerase to assemble a new strand that is the desired product of the entire polymerase chain reaction. The last stage, stage three, puts the samples through one cycle of 72⁰C for 3 minutes. There is a final hold of 4⁰C that preserves the DNA. The samples were then taken out of the PCR machine. The target sequence had been amplified a million times and could now be analyzed with less sensitive equipment! To analyze the sequence, see the Fluorimeter section.


Fluorimeter Measurements

To analyze the DNA samples after they've been amplified, a smartphone with adjustable settings was used.
These settings were used and the provided cradle was implemented to hold the phone in position for photographing:

- Exposure to highest setting
- ISO to 800+
- White balance to Automatic
- Saturation to highest setting
- Flash off if necessary
- Contrast to lowest setting


The Fluorimeter aparatus consists of a hydrophobic Teflon surface on a glass slide. There is an array of 3x10 glass wells on the surface to anchor drops in position for photographing. First, two drops of the cybergreen solution were placed on row 2, column 1&2 (Figure 1). With a sterile pipet two drops of amplified sample DNA were added to the cybergreen solution. It was important to place the DNA on top of the cybergreen in order to achieve the best results and to avoid contamination of the DNA samples. The picture was then photographed in the dark box with the door shut to eliminate excess background light. The next sample was prepared by sucking the liquid off the slide using a 'waste' pipet. The slide was then moved back by two columns for the next sample. This procedure was replicated for each of the patient samples and two slides were used in their entirety.

Picture and Analysis:
The pictures of each sample were analyzed using the "image j" software to process the images and analyze their content. The pictures, taken with the settings listed above, were upload to the "image j" software. The images were then separated into their composite colors, red, blue, and green. The green portion of the images were then analyzed using the software and its "INTDEN" was recorded.


Interpreting results:

The cybergreen soluiton allowed the amount of DNA in a sample to be determined by its "INTDEN". The cybergreen glowed green in the presence of DNA and while this glow was not visable to the naked eye, the smart phone in conjunction with "image j" was able to quantify the luminosity in terms of the "INTDEN". The brighter samples contained more DNA and had a higher "INTDEN" value. This indicated that the sample reacted with the primers better and was cancerous.

Research and Development

Specific Cancer Marker Detection - The Underlying Technology

Polymerase Chain Reaction, or PCR, is used to ampllify a specific segment of DNA, in this case the segment known to code for cancer. A primer, which is an artifically synthesized piece of complementary DNA, binds to the desired DNA segment, and the enzyme taq polymerase catalyzes the replication of the DNA strand using dNTPs (free bases). This is repeated over and over to produce many copies of the DNA. Once the copies are made, a fluorescent dye that only bonds to DNA double strands is added to the solution. If a solution that shows fluorescence by using a fluorimeter, then that DNA sample contains the cancer-associated sequence.

The single nucleotide polymorphism, or SNP, that is linked to cancer is rs17879961. The DNA base sequence associated with cancer is ACT, while the non-cancer sequence is ATT. PCR works to identify ACT from ATT because the primers, which are complementary to DNA strands containing cancer-positive ACT, will not bind to DNA containing ATT, and instead of DNA double strands, the solution will only contain single strands. The fluorescent dye will only bind to double strands and will identify those.



Adapted from: http://users.ugent.be/~avierstr/principles/pcr.html



Results

Image:fluorimeterexcel.jpg


These Images were taken using a HTC evo4G using the settings described under flourimeter measurements.

Water and SYBR green

Image:fluorimeterarray.jpg

Water and SYBR green under split color conditions for green Image:WATERSYBR.jpg

SYBR green and Calf Thymus

Image:CalfThumus.jpg


Sample Integrated Density DNA μg/mL Conclusion
PCR: Negative Control E6 F6 G6
PCR: Positive Control E7 F7 G7
PCR: Patient 1 ID #####, rep 1 E8 F8 G8
PCR: Patient 1 ID #####, rep 2 E9 F9 G9
PCR: Patient 1 ID #####, rep 3 E10 F10 G10
PCR: Patient 2 ID #####, rep 1 E11 F11 G11
PCR: Patient 2 ID #####, rep 2 E12 F12 G12
PCR: Patient 2 ID #####, rep 3 E13 F13 G13


KEY

  • Sample =
  • Integrated Density = Using the imageJ software, the image was split into its 'red', 'green' and 'blue' color components. This value represents the amount of 'green' light measured from the excitation through the blue LED light shining through the sample and subtracted from the background value, which was 'black'
  • DNA μg/mL =
  • Conclusion =
Personal tools