BME100 f2018:Group1 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 Domenik Valdivia Role(s) Maddie Willbrandt Role(s) Chelsea Powles Role(s) Joseph Alonzo Role(s) Cristal Tijerino 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 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 Cup for discarded tips Micropipettor OpenPRC machine: shared by two groups PCR Reaction Sample List Tube Label PCR Reaction Sample Patient ID G1 + Positive control 64002 G1 - Negative control 85406 G1 1-1 Patient 1, replicate 1 G1 1-2 Patient 1, replicate 2 G1 1-3 Patient 1, replicate 3 G1 2-1 Patient 2, replicate 1 G1 2-2 Patient 2, replicate 2 G1 2-3 Patient 2, replicate 3 DNA Sample Set-up Procedure Put extracted DNA into a PCR tube Add primer 1 to the PCR tube Next, add primer 2 to the PCR tube Add nucleotides to the PCR tube Add polymerase to the tube Place PCR tube into the DNA thermal cycler (for multiple hours) OpenPCR program HEATED LID: 100°C INITIAL STEPS: 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 2 minutes FINAL STEP: 72°C for 2 minutes FINAL HOLD: 4°C Research and Development PCR - The Underlying Technology Component Function of a PCR Reaction For a PCR reaction, template DNA is needed so that copies can be made for research purposes. Primers help focus on what part of the DNA needs to be amplified and allows a nucleotide to be added. Polymerase is used to create new strands of DNA from the existing strands by reading the DNA code and attaching the correct nucleotides. Taq polymerase is specifically important for this reaction because it can withstand the heat in the thermal cycler. In a PCR reaction dNTP's are the building blocks of the reaction allowing millions of copies to be copied. Thermal Cycling In the initial step, the temperature changes multiple times for corresponding durations. First, the DNA double helix separates creating two single stranded DNA molecules -- this occurs at 95°C for 2 minutes. Second, the DNA denatures for 30 seconds at 95°C. Lastly, annealing occurs, this where the single stranded DNA molecules pair up, however, primers must lock onto their targets before the strands can rejoin. Next, the DNA Polymerase activates, during this, a primer attached to a single DNA strand begins to add complementary nucleotides onto the strand, this continues until it gets to the end of the strand and falls off -- this occurs at a temperature of 72°C for 30 seconds. The second to final part of the cycling occurs at a temperature of 72°C for a 2 minute period, during this time the DNA Polymerase copies strands. After this there is a final hold that occurs at a temperature of 4°C, this maintains the DNA, the cycle is now over. Base Anneals A anneals to T T anneals to A G anneals to C C anneals to G Base-Pairing The first step of base pairing is when thermal cycler cools down to 50 degrees Celsius. Single stranded DNA molecules naturally attempt to pair up, but there are many more primer sequences than DNA strands in the tube. The primer strands will latch onto their target on the DNA before the single strands can rejoin. Next, the thermal cycler now heats up to 72 degrees Celsius, causing the DNA polymerase to activate. When DNA polymerase locates a primer attached to a single DNA strand, it begins to add complementary nucleotides onto the strand. It continues until it gets to the end of the strand and falls off. Source: https://bio.libretexts.org/@api/deki/files/11070/Biochemistry_Page_883_Image_0003.jpg SNP Information & Primer Design Background: About the Disease SNP A nucleotide is an essential building block for DNA and RNA. Each nucleotide must have a corresponding base such as: adenine, thymine, guanine or cytosine along with a molecule of sugar and phosphoric acid. A polymorphism is a regular and simultaneous occurrence in a single interbreeding population of two or more discontinuous genotypes. This variation is found in the Homo Sapiens species, it is located on the chromosome 12:40315266. This being said, the clinical significance of this SNP is uncertain, but, research associated with it points to Parkinson's Disease as being a linked condition. Leucine-rich repeat kinase 2 can be abbreviated by using "LRRK2". Three of its main functions are -- actin binding, ion channel binding, and protein binding. Alleles are mutations found at the same place on a chromosome there are usually one or more forms of this mutation. The disease associated allele contains the GAG codon, and the numerical position of the SNP is 40315266. Primer Design and Testing After completing all the steps in the primer test, we found that our primers were correct and received a 220bp sequence from the chromosome rs721710. After this, we repeated part a with the "Disease specific primers", and found "no matches", indicating that our work was correct. This should return no matches because a healthy individual would not have the base pair sequence.