BME103:W930 Group1 l2

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<!--- Include an illustration that shows how your system's primers allow specific amplification of the disease-related SNP ---> http://www.google.com/imgres?q=specific+amplification+of+heterotaxy&um=1&hl=en&client=safari&sa=N&tbo=d&rls=en&biw=1206&bih=684&tbm=isch&tbnid=3QBwBiTFWqfSZM:&imgrefurl=http://www.ipej.org/0802S/sreeram.htm&docid=7LM9Ij0u5jwLgM&imgurl=http://www.ipej.org/0802S/sreeram3.jpg&w=500&h=590&ei=tp2yUIuGDoPniwKmr4HYCw&zoom=1&iact=rc&dur=445&sig=104840076601863359039&page=1&tbnh=128&tbnw=121&start=0&ndsp=26&ved=1t:429,r:4,s:0,i:99&tx=59&ty=47
<!--- Include an illustration that shows how your system's primers allow specific amplification of the disease-related SNP ---> http://www.google.com/imgres?q=specific+amplification+of+heterotaxy&um=1&hl=en&client=safari&sa=N&tbo=d&rls=en&biw=1206&bih=684&tbm=isch&tbnid=3QBwBiTFWqfSZM:&imgrefurl=http://www.ipej.org/0802S/sreeram.htm&docid=7LM9Ij0u5jwLgM&imgurl=http://www.ipej.org/0802S/sreeram3.jpg&w=500&h=590&ei=tp2yUIuGDoPniwKmr4HYCw&zoom=1&iact=rc&dur=445&sig=104840076601863359039&page=1&tbnh=128&tbnw=121&start=0&ndsp=26&ved=1t:429,r:4,s:0,i:99&tx=59&ty=47
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[[Image:BME103_Group1_Heterotaxy_primer.png‎|200px|This represents the primers binding to the diseased '''A''' gene in a gene sequence that expresses the heterotaxy disease.]]
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Revision as of 13:23, 27 November 2012

BME 103 Fall 2012 Home
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Lab Write-Up 1
Lab Write-Up 2
Lab Write-Up 3
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Contents

OUR TEAM

Name: Kevin ChuExperimemtal Protocol Planner
Name: Kevin Chu
Experimemtal Protocol Planner
Name: StudentRole(s)
Name: Student
Role(s)
Name: StudentRole(s)
Name: Student
Role(s)
Name: StudentRole(s)
Name: Student
Role(s)
Name: StudentRole(s)
Name: Student
Role(s)
Name: StudentRole(s)
Name: Student
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Name: StudentRole(s)
Name: Student
Role(s)

LAB 2 WRITE-UP

Thermal Cycler Engineering

Our re-design is based upon the Open PCR system originally designed by Josh Perfetto and Tito Jankowski.


System Design


Key Features


Instructions





Protocols

Materials

Supplied in the Kit Amount
PCR Machine1
Fluorimeter1
DNA Solution6 samples (600.0μL)
Positive Control Solution1 sample (100.0μL)
Negative Control Solution1 sample (100.0μL)
Transfer Pipettes8
Tubes8
0.025% Tris BufferTBD

Each DNA solution consists of

DNA Solution Component Amount
Patient’s Template DNA*0.2μL
10μM forward primer1.0μL
10μM reverse primer1.0μL
GoTaq master mix50.0μL
dH2O47.8μL
Total100.0μL

  • Note that that positive control is calf thymus DNA, and the negative control is a blank solution of water.
Supplied by the User Amount
Smartphone1
Computer1
Open PCR Software1
ImageJ Software1
Sharpie (fine point)1

PCR Protocol

1. On your computer, download the Open PCR software.
2. After turning on the Open PCR machine, plug it into an electrical outlet by using the power adapter provided in the kit.
3. Connect the Open PCR machine to your computer by using the USB 2.0 cable.
4. Create a new program on OpenPCR by clicking on “Add a new experiment.”
a. Click on the “more options” button.
b. Click on the plus symbol next to initial step, and type 95 degrees Celsius for temperature and 180 seconds for time.
c. In the third section, type in 30 for the number of cycles.
d. Set the denaturing temperature to 95 degrees Celsius and time to 30 seconds.
e. Set the annealing temperature to 57 degrees Celsius and time to 30 seconds.
f. Set the extending temperature to 72 degrees Celsius and time to 30 seconds.
g. Add a final step. Set the temperature to 72 degrees Celsius and the time to 180 seconds.
h. Set the final hold to 4 degrees Celsius.
5. Use one pipette to move one of the extracted DNA samples into one of the PCR test tubes.
6. Next, add the forward and reverse primers to the test tube, using different pipettes for each.
7. Then, use another pipette to transfer GoTaq master mix into the DNA/primer mixture.
8. Dilute the contents of the test tube by filling its remainder with deionized water.
9. Repeat steps 5-8 for each of the seven remaining DNA samples including the positive and negative controls using different pipettes to transfer each substance.
10. Place the 8 test tubes including the positive and negative controls into the Open PCR machine, and close the lid and tighten the screw so that it barely touches the tops of the tubes.
11. Click on “Plug in Open PCR to start” to begin amplifying DNA samples.
12. Carefully observe the display screen on the lid of the Open PCR with the quantities that appear on the computer.
a. If these quantities are close, then continue running the program.
b. If these quantities are not close, then discontinue the program and try again.


DNA Measurement Protocol

Research and Development

Background on Disease Markers

Heterotaxty, (Hetero-different) (taxy-arrangement), syndrome is the most common birth defect that primary occurs in the heart. This syndrome is caused by the mutated gene, ZIC3. The reference number for this syndrome is rs104894962. This disease can also occur in other organs but it is less likely. With this syndrome, organs that are paired together have a mirror image of each other instead of having their own charcterstics. Other organs can also be arranged in a different order requiring major surgeries to aline the organs correctly. In some cases, organs or body parts may work incorrectly causing irregularity, worse infections, more recovery time, or lack of functioning correctly. This is not the only kind of the Heteotaxy however. As previously stated, the more common defects are located in the heart. Since most of the defects occur at birth, there is a varying type and severity. When the syndrome involves the heart, it is mainly because the heart sits to the right side of the chest instead of the left side.

Web Link - http://www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi?rs=104894962



Primer Design

The sequence for the heterotaxy disease allele is CCTACACGCACCCGAGCTCCCTGCGC [A/G] AACACATGAAGGTAATTACCCCTTT, with the mutation occurring at the [A/G] site. When the A gene is expressed, the mutation occurs and heterotaxy is coded. When the G gene is expressed, there is no mutation and the gene expression is normal.

Forward primer sequence (position 136,651,203 – 136,651,223, read left-right): TCCCTGCGCAAACACATGAA

Reverse primer sequence (200 base pairs to the right, read right-left): TCCCAACTTTGCTCACTCCC

A heterotaxy disease allele will show a PCR product because the disease allele will be amplified many times through the course of the chain reaction. Because a non-disease allele will not have a mutated expression of the A gene, it will not yield a PCR product and will instead amplify the healthy allele expression.


Illustration

http://www.google.com/imgres?q=specific+amplification+of+heterotaxy&um=1&hl=en&client=safari&sa=N&tbo=d&rls=en&biw=1206&bih=684&tbm=isch&tbnid=3QBwBiTFWqfSZM:&imgrefurl=http://www.ipej.org/0802S/sreeram.htm&docid=7LM9Ij0u5jwLgM&imgurl=http://www.ipej.org/0802S/sreeram3.jpg&w=500&h=590&ei=tp2yUIuGDoPniwKmr4HYCw&zoom=1&iact=rc&dur=445&sig=104840076601863359039&page=1&tbnh=128&tbnw=121&start=0&ndsp=26&ved=1t:429,r:4,s:0,i:99&tx=59&ty=47

This represents the primers binding to the diseased A gene in a gene sequence that expresses the heterotaxy disease.

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