BME103 s2013:T900 Group2

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(OUR TEAM)
Current revision (14:24, 26 March 2013) (view source)
(LAB 1 WRITE-UP)
 
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{| style="wikitable" width="700px"
{| style="wikitable" width="700px"
|- valign="top"
|- valign="top"
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| [[Image:BME103student.jpg|100px|thumb|Andy Son<br>Protocol Planner]]
+
| [[Image:Funny-monkey-05.jpg|100px|thumb|Andy Son<br>Protocol Planner]]
| [[Image:BME103student.jpg|100px|thumb|William Scott<br> R&D Scientist]]
| [[Image:BME103student.jpg|100px|thumb|William Scott<br> R&D Scientist]]
-
| [[Image:BME103student.jpg|100px|thumb|Name: student<br>Role(s)]]
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| [[Image:BME103student.jpg|100px|thumb|Joe Sasnone<br> R&D Scientist]]
-
| [[Image:BME103student.jpg|100px|thumb|Name: student<br>Role(s)]]
+
| [[Image:404005_4622115081677_1186647743_n.jpg|100px|thumb|Mitch Riggs <br> Open PCR Machine Engineer]]
| [[Image:BME103student.jpg|100px|thumb|Name: student<br>Role(s)]]
| [[Image:BME103student.jpg|100px|thumb|Name: student<br>Role(s)]]
| [[Image:BME103student.jpg|100px|thumb|Name: student<br>Role(s)]]
| [[Image:BME103student.jpg|100px|thumb|Name: student<br>Role(s)]]
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'''The Original Design'''<br>
'''The Original Design'''<br>
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(Add image of the full OpenPCR machine here, from the Week 9 exercise. Write a paragraph description for visitors who have no idea what this is)<br>
+
[[Image:0319131028.jpg]] <br>
-
 
+
The Open PCR machine is a small wooden box used to copy DNA. It achieves this by going through several cycles of heating and cooling. The length, temperature, and number of cycles can be changed with a computer hooked up to the device via USB.
'''Experimenting With the Connections'''<br>
'''Experimenting With the Connections'''<br>
-
When we unplugged (part 3) from (part 6), the machine ... (did what? fill in your answer)
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When we unplugged (part 3) from (part 6), the screen turned off. <br>
-
When we unplugged the white wire that connects (part 6) to (part 2), the machine ... (did what? fill in your answer)
+
When we unplugged the white wire that connects (part 6) to (part 2), the screen showed the wrong temperature. <br>
'''Test Run'''
'''Test Run'''
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(Write the date you first tested Open PCR and your experience(s) with the machine)<br>
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We first ran the machine on Tuesday, March 5.  We used unit 3.  We performed a simple test with the computer.  The temperatures shown on the computer corresponded with the temperatures shown on the device indicating a successful test.<br>
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'''Thermal Cycler Program'''<br>
'''Thermal Cycler Program'''<br>
 +
The set-up used for the program is as follows<br>
 +
Stage one: 1 cycle, 95 degrees Celsius for 3 minutes<br>
 +
Stage two: 35 cycles, 95 degrees Celsius for 30 seconds, 57 degrees Celsius for 30 seconds, 72 degrees Celsius for 30 seconds<br>
 +
Stage three: 72 degrees Celsius for 3 minutes <br>
 +
Final Hold: 4 degrees Celsius
'''DNA Sample Set-up'''<br>
'''DNA Sample Set-up'''<br>
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{| {{table}}
{| {{table}}
|-
|-
-
| row 1 cell 1 || row 1 cell 2 || row 1 cell 3 || row 1 cell 4
+
| Positive control:<br>cancer DNA template<br> Tube label: 1 || Patient 1<br>ID: 91862<br> Replicate 1<br> Tube label: 2 || Patient 1<br> ID: 91862<br> Replicate 2<br> Tube label: 3 || Patient 1<br> ID: 91862<br> Replicate 3<br> Tube label: 4
|-
|-
-
| row 2 cell 1 || row 2 cell 2 || row 2 cell 3 || row 2 cell 4
+
| Negative Control: no<br> DNA template<br> Tube label: 5 || Patient 2<br>ID: 28235<br> Replicate 1<br> Tube label:6 || Patient 2 <br> ID: 28235<br> Replicate 2<br> Tube label: 7 || Patient 2<br> ID: 28235<br> Replicate 3<br> Tube label: =)
|}
|}
'''DNA Sample Set-up Procedure'''
'''DNA Sample Set-up Procedure'''
-
# Step 1
+
# Step 1: First gather the necessary materials to set-up the DNA samples (pipette, PCR reaction mix, 8 transfer pipettes)
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# Step 2
+
# Step 2: Set the pipette to 50 microliters
-
# Step 3...
+
# Step 3: Place the transfer pipette onto the pipette to prevent cross contamination (never re-use).
 +
# Step 4: Use the pipette to transfer 50 microliters of each tube in the PCR reaction mix and transfer accordingly to the DNA sample tubes corresponding to the labels.
 +
# Step 5: Place the set of mixed tubes into the Open PCR machine and shut it tightly.
 +
# Step 6: Hook up the machine to a computer and run the Open PCR application with the pre set-up Thermal Cycler program.
 +
# Step 7: Once the application is done running, take out the tubes and hand it to the professor.
'''PCR Reaction Mix'''
'''PCR Reaction Mix'''
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* What is in the PCR reaction mix?
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* The PCR reaction mix contains 8 tubes of 50 microliter samples that contain a mix of Taq DNA polymerase, MgCl2, dNTP's, forward primers, and reverse primers.
'''DNA/ primer mix'''
'''DNA/ primer mix'''
-
* What is in the DNA/ primer mix?
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* The DNA/primer mix consists of samples of DNA of various patients. DNA/ primer mix contains 8 tubes, 50 μL each: Each mix contains a different template DNA. All tubes have the same forward primer and reverse primer.
 +
<br><br>
 +
==Research and Development==
 +
'''Specific Cancer Marker Detection - The Underlying Technology'''<br>
 +
Polymerase Chain Reaction (PCR) is a scientific method that utilizes DNA Polymerase to create a complimentary base strand from a template strand of DNA. Triphosphate nucleotides align with open DNA strands and DNA polymerase works to link the complementary nucleotide bases together growing strands through both condensation and hydroysis reactions. One major issue with DNA polymerase is that DNA strands are anti parallel, and polymerase is only able to add nucleotides to the free 3'OH group hence it can only build new strands in the 5'-3' direction (Sadava 279). Therefore, to correct for this issue the presence of a primer is required so that polymerase can proceed with directing the new nucleotides in place. Through these mechanisms it is possible to target specific positions on the template DNA sequence that a scientist intends to amplify(PCR 1). When the PCR process is completed the targeted DNA sequence containing the single-nucleotide polymorphism (SNP) will have manufactured over a billion copies (amplicons).
-
<br><br>
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The SNP included in the amplicon for this experiment was  denoted as rs17879961. This polymorphism is a variant of the CHEK2 gene which if present in a person's genome may increase the risk of developing breast cancer (Brennan et al 1795). This SNP is signified by a single base change from a Thymine (T) to a Cytosine (C) located on chromosome 22. With the PCR system, forward and reverse primers can be designed to target the cancer gene abnormality and amplify/multiply it. The normal DNA strands may multiply, but not as exponentially as the abnormal cancer DNA strands.
-
==Research and Development==
+
<div class="slide">
-
'''Specific Cancer Marker Detection - The Underlying Technology'''<br>
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Sources:
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(Add a write-up of the information discussed in Week 3's class)<br>
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Brennan, Paul et al. Uncommon CHEK2 Mis-sense Variant and Reduced Risk of Tobacco-related Cancers: Case-control Study. Rep. 15th ed. Vol. 16. Oxford: Advance Access, 2007. Human Molecular Genetics. Oxford University Press, 21 May 2007. Web. 25 Mar. 2013. PDF. <http://hmg.oxfordjournals.org/content/16/15/1794.full.pdf>.
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(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.)
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"PCR Introduction." NCBI. U.S. National Library of Medicine, n.d. Web. 25 Mar. 2013. <http://www.ncbi.nlm.nih.gov/projects/genome/probe/doc/TechPCR.shtml>.
 +
 
 +
Sadava, David E. Life: The Science of Biology. Sunderland, MA: Sinauer Associates, 2011. Print.
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Current revision

BME 103 Spring 2013 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

Andy SonProtocol Planner
Andy Son
Protocol Planner
William Scott R&D Scientist
William Scott
R&D Scientist
Joe Sasnone R&D Scientist
Joe Sasnone
R&D Scientist
Mitch Riggs  Open PCR Machine Engineer
Mitch Riggs
Open PCR Machine Engineer
Name: studentRole(s)
Name: student
Role(s)
Name: studentRole(s)
Name: student
Role(s)

LAB 1 WRITE-UP

Initial Machine Testing

The Original Design
Image:0319131028.jpg
The Open PCR machine is a small wooden box used to copy DNA. It achieves this by going through several cycles of heating and cooling. The length, temperature, and number of cycles can be changed with a computer hooked up to the device via USB.

Experimenting With the Connections

When we unplugged (part 3) from (part 6), the screen turned off.

When we unplugged the white wire that connects (part 6) to (part 2), the screen showed the wrong temperature.


Test Run

We first ran the machine on Tuesday, March 5. We used unit 3. We performed a simple test with the computer. The temperatures shown on the computer corresponded with the temperatures shown on the device indicating a successful test.




Protocols

Thermal Cycler Program
The set-up used for the program is as follows
Stage one: 1 cycle, 95 degrees Celsius for 3 minutes
Stage two: 35 cycles, 95 degrees Celsius for 30 seconds, 57 degrees Celsius for 30 seconds, 72 degrees Celsius for 30 seconds
Stage three: 72 degrees Celsius for 3 minutes
Final Hold: 4 degrees Celsius

DNA Sample Set-up

Positive control:
cancer DNA template
Tube label: 1
Patient 1
ID: 91862
Replicate 1
Tube label: 2
Patient 1
ID: 91862
Replicate 2
Tube label: 3
Patient 1
ID: 91862
Replicate 3
Tube label: 4
Negative Control: no
DNA template
Tube label: 5
Patient 2
ID: 28235
Replicate 1
Tube label:6
Patient 2
ID: 28235
Replicate 2
Tube label: 7
Patient 2
ID: 28235
Replicate 3
Tube label: =)

DNA Sample Set-up Procedure

  1. Step 1: First gather the necessary materials to set-up the DNA samples (pipette, PCR reaction mix, 8 transfer pipettes)
  2. Step 2: Set the pipette to 50 microliters
  3. Step 3: Place the transfer pipette onto the pipette to prevent cross contamination (never re-use).
  4. Step 4: Use the pipette to transfer 50 microliters of each tube in the PCR reaction mix and transfer accordingly to the DNA sample tubes corresponding to the labels.
  5. Step 5: Place the set of mixed tubes into the Open PCR machine and shut it tightly.
  6. Step 6: Hook up the machine to a computer and run the Open PCR application with the pre set-up Thermal Cycler program.
  7. Step 7: Once the application is done running, take out the tubes and hand it to the professor.

PCR Reaction Mix

  • The PCR reaction mix contains 8 tubes of 50 microliter samples that contain a mix of Taq DNA polymerase, MgCl2, dNTP's, forward primers, and reverse primers.

DNA/ primer mix

  • The DNA/primer mix consists of samples of DNA of various patients. DNA/ primer mix contains 8 tubes, 50 μL each: Each mix contains a different template DNA. All tubes have the same forward primer and reverse primer.




Research and Development

Specific Cancer Marker Detection - The Underlying Technology

Polymerase Chain Reaction (PCR) is a scientific method that utilizes DNA Polymerase to create a complimentary base strand from a template strand of DNA. Triphosphate nucleotides align with open DNA strands and DNA polymerase works to link the complementary nucleotide bases together growing strands through both condensation and hydroysis reactions. One major issue with DNA polymerase is that DNA strands are anti parallel, and polymerase is only able to add nucleotides to the free 3'OH group hence it can only build new strands in the 5'-3' direction (Sadava 279). Therefore, to correct for this issue the presence of a primer is required so that polymerase can proceed with directing the new nucleotides in place. Through these mechanisms it is possible to target specific positions on the template DNA sequence that a scientist intends to amplify(PCR 1). When the PCR process is completed the targeted DNA sequence containing the single-nucleotide polymorphism (SNP) will have manufactured over a billion copies (amplicons).

The SNP included in the amplicon for this experiment was denoted as rs17879961. This polymorphism is a variant of the CHEK2 gene which if present in a person's genome may increase the risk of developing breast cancer (Brennan et al 1795). This SNP is signified by a single base change from a Thymine (T) to a Cytosine (C) located on chromosome 22. With the PCR system, forward and reverse primers can be designed to target the cancer gene abnormality and amplify/multiply it. The normal DNA strands may multiply, but not as exponentially as the abnormal cancer DNA strands.

Sources:

Brennan, Paul et al. Uncommon CHEK2 Mis-sense Variant and Reduced Risk of Tobacco-related Cancers: Case-control Study. Rep. 15th ed. Vol. 16. Oxford: Advance Access, 2007. Human Molecular Genetics. Oxford University Press, 21 May 2007. Web. 25 Mar. 2013. PDF. <http://hmg.oxfordjournals.org/content/16/15/1794.full.pdf>.

"PCR Introduction." NCBI. U.S. National Library of Medicine, n.d. Web. 25 Mar. 2013. <http://www.ncbi.nlm.nih.gov/projects/genome/probe/doc/TechPCR.shtml>.

Sadava, David E. Life: The Science of Biology. Sunderland, MA: Sinauer Associates, 2011. Print.





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