BME100 f2018:Group13 T0800 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: Derek Busch Role(s): Editor Name: Gabriel Baca Role(s): Protocol Name: Mia Geric Role(s): Protocol Name: Michelle Tieu Role(s): R&D Name: Damian Gordillo-Velazquez Role(s): SNP LAB 4 WRITE-UP Protocol Materials Lab Coat Disposable Gloves PCR Reaction Mix, 8 tubes, 50 μl each:Mix contains Taq DNA polymerase, MgCl_2, and dNTP's (hp://www.promega.com/resources/protocols/product‐informaon‐sheets/g/gotaq‐colorless ‐master‐mix‐m714‐protocol/) DNA/ primer mix, 8 tubes, 50 μL each: Each mix contains a different template DNA. All tubes have the same forward primer and reverse primer A strip of empty PCR tubes Disposable pipette tips: only use each only once. Never reuse disposable pipette tips. If you do, the samples will become cross-contaminated. Cup for discarded tips Micropipettor OpenPCR Machine: shared by two groups PCR Reaction Sample List Tube Label PCR Reaction Sample Patient ID G13 + Positive control none G13 - Negative control none G13 1-1 Patient 1, replicate 1 81477 G13 1-2 Patient 1, replicate 2 81477 G13 1-3 Patient 1, replicate 3 81477 G13 2-1 Patient 2, replicate 1 68313 G13 2-2 Patient 2, replicate 2 68313 G13 2-3 Patient 2, replicate 3 68313 DNA Sample Set-up Procedure Step 1: Move the extracted DNA into the PCR tube Step 2: Add primer 1 to the PCR tube Step 3: Add primer 2 to the PCR tube Step 4: Add nucleotides to the PCR tube Step 5: Add DNA polymerase to the PCR tube Step 6: Place the PCR tube in the thermal chamber OpenPCR program Heated Lid 100°C Initial Step 95°C for 2 minutes Number of cycles:25 (Denature at 95°C for 30 seconds, and Extend at 72°C for 30 seconds) Final step: 72°C for 30 seconds Final hold: 4°C Research and Development PCR - The Underlying Technology Q1. What is the function of each component of a PCR reaction? * Template DNA: It is the specific part of the DNA that will be copied, it is where you extracting the cells in order to make more copies. * Primers: Two primers are specifically designed so they are able to match to a segment of DNA. * Taq Polymerase: Reads the DNA code and attaches them to the matching nucleotides to create DNA copies, and the specific Thermus Aquaticus Polymerase is able to withstand the high temperatures of the reaction. * Deoxyribonucleotides (dNTP's): They are the building blocks for DNA replication where the DNA polymerase will match up the correct base pairs for the new DNA according to the original strand’s sequence. Q2. What happens to the components (listed above) during each step of thermal cycling? * Initial Step: 95°C for 2 minutes: Double-stranded DNA is separated into single strands. * Denature at 95°C for 30 seconds: Hydrogen bonds between DNA strands break. * Anneal at 57°C for 30 seconds: Primers are attached to the DNA, one for each strand. * Extend at 72°C for 30 seconds: DNA polymerase binds to the primers and begins to add nucleotides to the DNA strand. * Final Step: 72°C for 2 minutes: The complementary strand is is built by the DNA polymerase. * Final Hold: 4°C: Holding the DNA samples at this temperature prevents sample loss and degradation. Q3. DNA is made up of four types of molecules called nucleotides, designated as A, T, C, and G. Base-pairing, driven by hydrogen bonding, allows base-pairs to stick together. Which base anneals to each base listed below? If you need help, use the "Build a DNA Molecule Tool". * Adenine (A): Thymine (T) * Thymine (T): Adenine (A) * Cytosine (C): Guanine (G) * Guanine (G): Cytosine (C) Q4. During which two steps of thermal cycling does base-pairing occur? Explain your answers. * Base pairing occurs during the extending stage when the sample is held at 72°C for 30 seconds. At this stage, the DNA taq polymerase adds base pairs to the template DNA one by one from the 5’ end to the 3’ end of the strand. SNP Information & Primer Design Background: About the Disease SNP The single nucleotide polymorphism is Leucine Rich Repeat Kinase 2 (LRRK2) , which is found in humans, on the 12:40315266 chromosome. LRRK2 encodes a protein with an akryin repeat region, a leucine-rich repeat domain, a kinase domain, a DFG-like motif, a RAS domain, a GTPase domain, a MLK-like domain, and a WD40 domain. It contains mutations that are linked to Parkinsonism. The disease-associated allele (one of two or more alternative forms of a gene that arise by mutation and are found at the same place on a chromosome), contains the CAG codon. The position of the SNP is 40315266. Primer Design and Testing In our primer test, we found that there were no matches because there was a mutation, which has changed the sequence, causing it to not be recognized.