BME100 f2018:Group1 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 Connor Rosenbaum Role(s) Joe Black Role(s) Quinten Hunter Role(s) May Nguyen Role(s) Natalie Stevens Role(s) LAB 4 WRITE-UP Protocol Materials Lab coat and disposable gloves PCR reaction mix, 8 tubes, 50 μL each: Mix contains Taq DNA polymerase, MgCl2, and dNTP’s (http://www.promega.com/resources/protocols/product‐information‐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 ps Micropipettor OpenPCR machine: shared by two groups PCR Reaction Sample List Tube Label PCR Reaction Sample Patient ID G1 + Positive control None G1 - Negative control None G1 1-1 Patient 1, replicate 1 26963 G1 1-2 Patient 1, replicate 2 26963 G1 1-3 Patient 1, replicate 3 26963 G1 2-1 Patient 2, replicate 1 29896 G1 2-2 Patient 2, replicate 2 29896 G1 2-3 Patient 2, replicate 3 29896 DNA Sample Set-up Procedure Acquire all listed materials. Cut PCR tubes so that there are two rows with four tubes each. With a marker, label the sides of the empty tubes with the Tube Labels listed above. Put the tubes on a rack. Begin with the empty postive-control labelled tube. Pipette 50 microliters of PCR reaction mix into the tube. Throw away the disposable tip. Using another tip, pipette the positive-control DNA mix into the same tube as before. The tube should now have 100 microliters in it. Repeat steps 5 and 6 for the next seven tubes (negative control, patient 1 replicates 1,2,3 , patient 2 replicates 1,2,3 ), using the corresponding DNA mixes for each tube. When finished, all tubes should have 100 microliters in them. Close the lids on the tubes tightly. Bring the tubes to the PCR machine. Put the tubes into the thermal cycler. Be sure to only run the machine when all 16 slots are filled; two group use the machine at once. 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 Component Functions in a PCR Reaction? Template DNA, primers, Taq polymerase, and deoxyribonucleotides (dNTP's) are all essential in a PCR reaction. Template DNA is a single strand of DNA that the DNA polymerase enzyme uses as a basis to copy the DNA. A primer is a short single strand of RNA or DNA that initiates DNA synthesis. This is necessary since the enzymes that catalyze DNA replication only add new nucleotides to already existing strands of DNA. Taq polymerase is an enzyme that can handle the high temperatures that would otherwise denature the proteins during the PCR reaction. In this reaction, it replaces DNA polymerase. Deoxyribonucleotides are monomers of DNA. Each one is made of up a nitrogenous base, a deoxyribose sugar, and one phosphate group. Steps of Thermal Cycling During the initial step (95°C for 2 minutes), the DNA strands begin unraveling. At the Denaturing step (95°C for 30 seconds), the DNA strands are separated into two individual strands. In the Annealing step (57°C for 30 seconds), the temperature is lowered so that the DNA primers can attach to the template DNA. During the Extending step (72°C for 30 seconds), a new DNA strand is created by the Taq polymerase enzyme. The final step (72°C for 2 minutes) is the optimal temperature for the Taq polymerase to build the complementary strand. The final hold (4°C) limits the activity of the Taq polymerase, in order to prevent non-specific binding of primers, so that the DNA is stable. Base Pairs The four types of nucleotides are Adenine, Thymine, Cytosine, and Guanine. Adenine and Thymine pair together, while Cytosine and Guanine pair together. Base-Pairing during Thermal Cycling Base-pairing occurs during the anneal and extend steps of thermal cycling. In the anneal step, the two individual strands are heated so the DNA primers can bind with the template DNA, and this is where the positions for replication is originated. Then, when the temperature is elevated slightly, and the extension step begins. In this step, two taq polymerase match the base pairs together from the primers to their pairs. With both of these steps combined, the base-pairing is able to be completed. SNP Information & Primer Design Background: About the Disease SNP SNP is single nucleotide polymorphism. A nucleotide is the basic structural unit and building block for DNA. There are four nucleotides which are Adenine, Guanine, Cytosine, Thymine. A polymorphism is two or more clearly different morphs or forms, also referred to as alternative phenotypes, in the population of a species. A variation of SNP, rs721710, is found in Homo Sapiens. It is located on chromosome 12:40315266. There is no clear clinical significance of this SNP, but it has been linked to Parkinson's disease. LRRK2 stands for leucine-rich repeat kinase 2. The functions of LRRK2 include ATP-Binding, GTP-Binding, and GTP-binding depnedent-protein kinase activity. An allele is an alternate form of a gene that arises from a mutation and is found in the same place on a chromosome. The disease-associated allele contains the codon GAG. The numerical position of the SNP is 40315266. Primer Design and Testing The non-disease forward primer (20nt) is 5' T T A A G T G A C T T G T A C T T T G T 3'. The numerical position exactly 200 bases to the right of the disease SNP is 40315466. The non-disease reverse primer is 5' T G A A G C T C T T C A A G T A G T C T 3'. The disease forward primer is 5' T T A A G T G A C T T G T A C T T T T A 3'. The disease reverse primer is 5' T G A A G C T C T T C A A G T A G T C T 3'. When using the UCSC In-Silico PCR website, we found that the non-disease forward primer and the non-disease reverse primer had a 220 bp. From this we can conclude that there weren't any issues or mutations in the sequence. In the disease forward primer and the reverse primer there was no match. From this we could assume that there was a possible mutation.