BME100 s2014:T Group10 L4
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LAB 4 WRITE-UP
Initial Machine Testing
The Original Design
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.
We first tested Open PCR on March 20, 2014. The heating protocol was as follows:
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.
Thermal Cycler Program
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.
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:
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:
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:
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.
Garcia, Tony, and Karmella Haynes. BME 100 Lab Workbook. N.p.: ASU BME 100, n.d. DOC.