BME100 f2015:Group2 8amL4

BME 100 Fall 2015	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: Bryce Goldthwaite Role(s) Name: Stephen Lane Role(s) Name: Samuel Rios Role(s) Name: Christian Manriquez Role(s) Name: Yash Dalbi Role(s) LAB 4 WRITE-UP Protocol Materials PPE PCR reaction mix, 8 tubes, 50 μL each: Mix contains Taq DNA polymerase, MgCl2, and dNTP’s DNA/ primer mix, 8 tubes, 50 μL each Disposable pipette tips Cup for discarded tips Micropipettor OpenPCR machine PCR Reaction Sample List Tube Label PCR Reaction Sample Patient ID G2 + Positive control none G2 - Negative control none G2 1-1 Patient 1, replicate 1 65991 G2 1-2 Patient 1, replicate 2 65991 G2 1-3 Patient 1, replicate 3 65991 G2 2-1 Patient 2, replicate 1 29837 G2 2-2 Patient 2, replicate 2 29837 G2 2-3 Patient 2, replicate 3 29837 DNA Sample Set-up Procedure Step 1: Move extracted DNA into the special PCR test tube Step 2: Add Primer 1 to the PCR test tube Step 3: Add Primer 2 to the PCR test tube Step 4: Add nucleotides to the PCR test tube Step 5: Add DNA polymerase to the PCR test tube Step 6: Place the PCR test tube in the thermal cycler OpenPCR program HEATED LID: 100°C INITIAL STEP: 95°C for 2 minutes NUMBER OF CYCLES: 25 Denature at 95°C for 30 seconds, Anneal at 57°C for 30 seconds, and extend at 72°C for 30 seconds FINAL STEP: 72°C for 2 minutes FINAL HOLD: 4°C Research and Development PCR - The Underlying Technology Components and Functions _________________________ Template DNA Template DNA is the target strand of DNA where the disease in question exists as a mutated base pair. Primers Primers are short segments of DNA that are synthesized in the lab. Primers consist of forward primers and reverse primers, each made of about 20 nucleotides that match with a unique segment of the template DNA. Primers attach to either end of the template DNA strand. They allow DNA Polymerase to attach to the DNA to begin replication. Taq Polymerase DNA Polymerase is the complex of proteins that is responsible for replicating a strand of DNA by matching base pairs of nucleotides with the template DNA. DNA Polymerase attaches to a segment of template DNA via primers, then runs along the DNA like a train track, pulling nucleotides from the surrounding environment to build a complimentary strand. The most frequently used polymerase is Taq Polymerase, which is taken from the Thermus aquaticus (thus, “T-aq”) bacteria, which lives in the hot springs of Yellowstone National Park. Taq polymerase is used because it can function at the high temperatures required for the PCR process without denaturing. Deoxyribonucleotides (dNTPs) Deoxyribonucleotides, or simply “nucleotides,” are the A’s (Adenine), T’s (Thymine), C’s (Cytosine), and G’s (Guanine) that code for the amino acids that make up proteins. Adenine matches to Thymine and Cytosine matches to Guanine via hydrogen bonds. In PCR, nucleotides are pulled from the surrounding environment by the Taq Polymerase and are matched to the template DNA strand in order to create a replica strand. The Steps of Thermal Cycling __________________________ Initial Step: 95°C for 3 minutes: This step heats the polymerase to prepare it for the PCR reaction. Denature ​at 95°C for 30 seconds: During this step, the hydrogen bonds joining the base pairs of nucleotides together in the template strand of DNA are separated, or "denatured." This separation sets the stage for the primers to bind to their respective strands in order for the PCR reaction to begin. Anneal ​at 57°C for 30 seconds: Now that the template DNA is separated into two strands, the primers can now bind to their target sites during this step. This temperature is just hot enough for the primers to behave as they should and bind to the DNA strands. Once the primers bind to the DNA, the Taq Polymeras binds to the primers. Extend ​at 72°C for 30 seconds: At this temperature, the PCR reaction begins. The Taq Polymerase runs along the single strands of DNA in the 5' to 3' direction and builds complimentary strands by pulling unbound nucleotides from the surrounding liquid environment. The Denature, Anneal, and Extend steps are then repeated, causing the target DNA strands to multiply exponentially. Final Step: 72°C for 3 minutes: This step insures that any remaining PCR reactions can be completed. Final Hold: 4°C: The replicated target DNA strands can now be stored for short-term use during this step. SNP Information & Primer Design Background: About the Disease SNP What is a nucleotide? A nucleotide is the structural unit or building block of a DNA molecule. What is a polymorphism? A polymorphism refers to the occurrence of two or more phenotypes within the same species. The single nucleotide polymorphism rs1805008 occurs in Homo sapiens. It can be found on the MC1R gene located on the 16:89919736 chromosome. The clinical significance of the rs1805008 of this SNP is that it is pathogenic. This gene, MC1R is associated with Parkinson disease and can increase susceptibility to clear cell renal cell carcinoma. Primer Design and Testing MC1R stands for melanocortin 1 receptor (alpha melanocyte stimulating hormone receptor). The MC1R gene provides instructions for making a protein called the melanocortin 1 receptor. The first three terms unique saw were G-protein coupled peptide receptor activity, melanocortin receptor activity, and melanocyte-stimulating hormone receptor activity. An allele is one of two or more alternative forms of a gene that arise by mutation and are found at the same place on a chromosome. The disease-associated allele contains the sequence TGG. The numerical sequence of the SNP is 89919736. The non-disease forward primer (20nt) is 5'-CAGCATCGTGACCCTGCCGC. The numerical position exactly 200 bases to the right of the disease SNP is 89919936. The non-disease reverse primer is 5'-CTTGTGGAGCCGGGCGATGC. The disease forward primer is 5'-CAGCATCGTGACCCTGCCGT and the disease reverse primer is 5'-CTTGTGGAGCCGGGCGATGC.