BME100 f2018:Group8 T1030 L4

BME 100 Fall 2018	Home People Lab Write-Up 1 | Lab Write-Up 2 | Lab Write-Up 3 Lab Write-Up 4 | Lab Write-Up 5 | Lab Write-Up 6 Course Logistics For Instructors Photos Wiki Editing Help
OUR TEAM Name: Sabrina Worley Role(s) Name: Ty Promreuk Role(s) Name: Sonja Brett Role(s) Name: Jaad Waters Role(s) LAB 4 WRITE-UP Protocol Materials Lab coat and disposable gloves PCR reaction mix (Taq DNA polymerase, MgCl2, and dNTPs), 8 tubes, 50 μL each DNA primer mix, 8 tubes, 50 μL each Strip of empty PCR tubes Disposable pipette tips Cup for discarded tips Micropipettor OpenPCR machine PCR Reaction Sample List Tube Label PCR Reaction Sample Patient ID G8 + Positive control none G8 - Negative control none G8 1-1 Patient 1, replicate 1 21105 G8 1-2 Patient 1, replicate 2 21105 G8 1-3 Patient 1, replicate 3 21105 G8 2-1 Patient 2, replicate 1 12210 G8 2-2 Patient 2, replicate 2 12210 G8 2-3 Patient 2, replicate 3 12210 DNA Sample Set-up Procedure Extract DNA from a sample of blood, hair, or saliva. Move the extracted DNA into the designated PCR tube. Add Primer 1 to the PCR tube. This will attach to one end of the separated strand. Add Primer 2 to the PCR tube. This will attach to the opposite end of the other single strand. Add a solution of nucleotides to the PCR tube. Add DNA polymerase to the PCR tube. These molecules read the code of the single strands and attach the loose nucleotides to their matching pair. Finally, place the PCR tube into the Thermal Cycler. OpenPCR program Heat the lid to 100°C. For 25 cycles: For initial step, heat to 95°C for 2 minutes. Denature at 95°C for 30 seconds. Anneal at 57°C for 30 seconds. Extend at 72°C for 30 seconds. Leave at 72°C for 2 minutes to perform final step (ensuring completion of the elongation process). Cool to 4°C to perform final hold. Research and Development PCR - The Underlying Technology What are the components in PCR? The components of a polymerase chain reaction are: the template DNA, DNA primers, Taq DNA polymerase enzyme, and deoxyribonucleotides (dNTPs). The template DNA acts as a template for the DNA to be replicated. Often, this DNA contains viral or bacterial infections, or some type of genetic variance. By replicating this template DNA, these variances are amplified and more easily observed. Primers are designed to match the segment of template DNA to be copied. One primer attaches to the top strand at one end of the DNA segment, and the other primer attaches to the bottom strand at the other end. The Taq polymerase is an enzyme which attaches to the primers and adds DNA nucleotides to extend the second strand. Deoxyribonucleotides, abbreviated as dNTPs, are the foundation of what composes DNA. The Taq polymerase enzyme attaches four types of nucleotides- A, T, C, and G- to the primers to create the replicated DNA strand. (Nucleotides attach, or anneal, to each other in complementary pairs: adenine (A) anneals to thymine (T), while cytosine (C) anneals to guanine (G).) Base pairing is how the primers attach to the template DNA, and how the nucleotides for the new DNA strand attach to the primers. How does PCR thermal cycling work? Thermal cycling in PCR is performed by a thermal cycling machine. PCR thermal cycling consists of three main stages: denaturing, or separating the double-stranded template DNA into single strands; annealing, or attaching primers to the template DNA; and extending, which employs the Taq DNA polymerase enzyme to create the new DNA strand. In denaturing, the temperature is raised to break the hydrogen bonds between the complementary base pairs, separating the two strands of the template DNA sample. The temperature is then lowered for the annealing process. "Annealing" refers to the process of the DNA primers attaching to each separated template DNA strand. For the final stage, extension, the temperature is raised to about 72°C and the Taq DNA polymerase enzyme binds to each primer and attaches DNA nucleotides, creating a new strand of DNA. Our "final step" holds the temperature of the extension stage for an additional 2 minutes to ensure that the process of extension completes fully on all strands of DNA. "Final hold" cools the system down to 4°C, ending the process. When does base pairing occur in thermal cycling? Base-pairing of nucleotides first occurs during the annealing stage, when the primers attach to the template DNA to prevent the separated strands from rejoining. Base-pairing is seen again during the extension stage, when the polymerase enzyme detects the primers and begins to attach the matching nucleotides onto the new strand of DNA. Original illustration: SNP Information & Primer Design Background: About the Disease SNP Single nucleotide polymorphisms, or SNPs, are the most common genetic variations among humans. Each SNP occurs within a single nucleotide in DNA, where the bases adenine, guanine, thymine, and cytosine are interchanged to cause the variations. Multiple nucleotide variations can occur at any specific position. These variations are alleles (a variant form of the gene). The different variations among humans are what causes the differences in susceptibility to diseases, and in the way that the body responds to treatment. Primer Design and Testing After designing the forward and reverse non-disease primers, we ran them through the non‐disease human genome sequence system in order to validate them. The non-disease primers passed the test with a result of 220 bp, meaning that the designed non-disease primers were accurate and did not contain any SNP variations. The disease specific primers were then run through the same system in order to validate them. The test resulted in no matches in the data base, due to the fact that it only tests for human gene sequences that do not contain the gene SNP. The forward and reverse disease primers designed were accurate and produced the results that were expected. Non-disease primer results: Disease specific primer results: