BME100 s2014:T Group10 L4

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Contents

OUR TEAM

Sean-Christian BradburyThursday Group 10 Member
Sean-Christian Bradbury
Thursday Group 10 Member
Suyen GoThursday Group 10 Member
Suyen Go
Thursday Group 10 Member
Nathaniel KirkpatrickThursday Group 10 Member
Nathaniel Kirkpatrick
Thursday Group 10 Member
Maya RobinsonThursday Group 10 Member
Maya Robinson
Thursday Group 10 Member
Lissette ValleThursday Group 10 Member
Lissette Valle
Thursday Group 10 Member

LAB 4 WRITE-UP

Initial Machine Testing

The Original Design


Open OpenPCR Machine from OpenPCR.org
(Figure 2: An Open OpenPCR Machine,
Original image from OpenPCR.org,
labels added by Nathan Kirkpatrick)


A PCR is a Polymerase Chain Reaction Machine that is used to replicate single or a few pieces of DNA into thousands to millions of identical DNA sequences. The machine uses heat to facilitate DNA melting, a process in which DNA strands are denatured, and cools it down so that Taq Polymerase (a DNA Polymerase that can function at high temperatures) can replicate the desired section/s of DNA. Essentially, a PCR machine amplifies DNA so that DNA sequence can be detected at a macroscopic level.

Experimenting With the Connections

When we unplugged the display screen from the motherboard, the machine's display screen ceased to function.

When we unplugged the white wire that connects motherboard to the face or PCR tube-holder plate, the machine's temperature display read with a temperature of -40 degrees Celsius. This led us to believe that the wire was the temperature sensor wire.

Test Run

We first tested Open PCR on March 20, 2014. The heating protocol was as follows:

  • Heated Lid: 100*C
  • Initial Step: 95*C for 2 minutes
  • Number of Cycles: 35
    • Denature: 95*C for 30 seconds
    • Anneal: 57*C for 30 seconds
    • Extend: 72*C for 30 seconds
  • Final Step: 72*C for 2 minutes
  • Final Hold: 4*C


Our Open PCR went through 12 cycles and proved it was fully functional. We marked our machine with "PASS" in response to this successful test run.

Our OpenPCR Machine with the PASS label
(Figure 3: Our Open PCR Machine with our Group 10 Pass Label)




Protocols

Thermal Cycler Program

  • Heated Lid: 100*C
  • Number of Cycles: 35
  1. Initial Step: 95*C for 3 minutes. DNA unwinds.
  2. Denature: 95*C for 30 seconds. Individual DNA strands separate.
  3. Anneal: 57*C for 30 seconds. Primers bind to complementary sites on the DNA strands.
  4. Extend: 72*C for 30 seconds. Taq Polymerase binds to the DNA strands and builds a new strand of nucleotides (dNTP's).
  5. Final Step: 72*C for 3 Minutes. Taq Polymerase builds a new strand of nucleotides.
  6. Final Hold: 4*C. This ensures that the nucleotides (dNTP's) are bound well to each other.


DNA Sample Set-up

Positive Control (PC) Patient 1, Replicate 1 (1R1) Patient 1, Replicate 2 (1R2) Patient 1, Replicate 3 (1R3)
Negative Control (NC) Patient 2, Replicate 1 (2R1) Patient 2, Replicate 2 (2R2) Patient 2, Replicate 3 (2R3)


DNA Sample Set-up Procedure

Micro-pipetting the PCR Mix
(Figure 4: Micropipette used to mix specific volumes of reagents for the PCR reaction)

  1. Step 1: Labeled each tube with the previously chosen codes (PC, NC, 1R1, 2R1, etc.)
  2. Step 2: Used the micropipette at 50 microliters to transfer the PCR reaction mix to the positive control tube. Repeated the process for the remaining 7 tubes, adding the appropriate replicates to their respective tubes and making sure to use a new tip for each mix.
  3. Step 3: Closed the lids on the reaction tubes.
  4. Step 4: Placed tubes in the PCR machine and began reaction.


PCR Reaction Mix

The PCR Reaction Mix contains Taq DNA Polymerase, MgCl2, and dNTP's. Taq DNA Polymerase is the enzyme that assembles nucleotides into new strands of DNA. MgCl2 is a molecule that is included to aid in the function of Taq DNA Polymerase. dNTP's are Deoxyribonucleotides which are the building blocks of DNA. They polymerize to form DNA.


DNA/ primer mix

A different template of DNA is in each DNA/ primer mix. However, the forward and reverse primers in all tubes are the same. The primers tell the Taq Polymerase where to start copying the DNA strand.





Research and Development

PCR - The Underlying Technology

Function of Components

Polymerase Chain Reaction Machines work by carefully controlling the temperature of DNA samples so the steps of the reaction can occur correctly. The reagents for PCR include:

  • Taq DNA Polymerase: the enzyme that assembles nucleotides into new strands of DNA
  • MgCl2: a molecule that is included to aid in the function of Taq DNA Polymerase
  • dNTP: Deoxyribonucleotides which are the building blocks of DNA and polymerize to form DNA
  • Forward Primer: binds to the 5' end of a particular sequence of a disease-associated allele
  • Reverse Primer: binds to the 3' end of a particular sequence of either the disease-associated allele or the non-disease-associated allele
  • DNA Sample: the DNA that is to be copied

PCR Steps

  1. Initial Step: 95*C for 3 minutes. DNA unwinds.
  2. Denature: 95*C for 30 seconds. Individual DNA strands separate.
  3. Anneal: 57*C for 30 seconds. Primers bind to complementary sites on the DNA strands.
  4. Extend: 72*C for 30 seconds. Taq Polymerase binds to the DNA strands and builds a new strand of nucleotides (dNTP's).
  5. Final Step: 72*C for 3 Minutes. Taq Polymerase builds a new strand of nucleotides.
  6. Final Hold: 4*C. This ensures that the nucleotides (dNTP's) are bound well to each other.

The process then repeats itself to create exponentially more copies of the target DNA.

NCBI Database Research on Disease-Associated Sequence rs237025

The National Center for Biotechnology (NCBI) has a tremendously helpful and informative website that contains information regarding numerous disease-associated genetic sequences for many types of organisms. Genetic sequences can cause diseases when small errors or variations occur in their chain of nucleotides. A nucleotide is an organic molecule that acts as a monomer in DNA and RNA. A type of error or variation that can occur in genetic sequences is called Single Nucleotide Polymorphism or SNP. A polymorphism is a common variation in the sequence of DNA among individuals. A variation occurring in more than 1% of the population is considered useful for genetic linkage analysis. (Wikipedia.org)

The SNP rs237025 is a genetic variation that occurs in Homo sapiens on chromosome 6. The clinical significance of this SNP is listed as "Other" on the NCBI dbSNP page, but PubMed revealed research linking this SNP to diseases such as Type 1 Diabetes, Vogt-Koyanagi-Harada Disease, as well as Type 2 Diabetes among others. (National Center for Biotechnology Information)

It is associated with genes SUM04 (387082) and TAB2 (23118). SUM04 stands for Small Ubiquitin-Like Modifier 4 in Homo sapiens. It functions on a molecular level by specifically modifying IKBA leading to negative regulation of NF-kappa-B-dependent transcription of the IL12B gene. (National Center for Biotechnology Information)

An allele is a different version of a gene that produces different effects. (Wikipedia.org) The disease-associated allele for rs237025 contains the sequence ATG. This sequence is located at position 149,721,690 of the SNP.

Designing a Sequence-Specific Primer Pair

The 20 nucleotide rs237025-specific forward primer that ends with the disease-associated allele has the following sequence:
5'-TGAACCACGGGGATTGTCAA-3'

The 20 nucleotide reverse primer starts at position 149,721,890 on the disease SNP. This primer is not specific to the disease sequence unlike the forward primer. It has the following sequence:
5'-TGTGGTGGAACCAAATTGCA-3'

The forward is designed to bind completely to a corresponding disease-SNP strand of DNA and the reverse will bind completely to its corresponding strand which could be either the disease-SNP strand, or the normal strand. Both primers are required to bind completely to DNA in order for PCR to work, so if the template had the non-disease allele, PCR would not occur. This is because, although the reverse primer would bind to the DNA, the forward primer would not bind completely.


Works Cited

Garcia, Tony, and Karmella Haynes. BME 100 Lab Workbook. N.p.: ASU BME 100, n.d. DOC.

OpenPCR Guts. Digital image. DNA Is Now DIY: OpenPCR Now Worldwide. OpenPCR.org, 6 July 2011. Web. 2 Apr. 2014.

"Polymorphism (biology)." Wikipedia. Wikimedia Foundation, 30 Mar. 2014. Web. 27 March 2014.

"PubMed Citations of Rs237025 SNP." National Center for Biotechnology Information. U.S. National Library of Medicine, n.d. Web. 27 March 2014.

"Reference SNP (refSNP) Cluster Report: Rs237025." NCBI. U.S. National Library of Medicine, n.d. Web. 27 March 2014.

"SUMO4 Small Ubiquitin-like Modifier 4." National Center for Biotechnology Information. U.S. National Library of Medicine, n.d. Web. 02 Apr. 2014.




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