BME100 f2018:Group3 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 Error creating thumbnail: File missing Name: Taylin Aasen Error creating thumbnail: File missing Name: Jaqueline Bello Mendoza Error creating thumbnail: File missing Joan Ponce Sanchez Error creating thumbnail: File missing Name: Kaprao Fuegner LAB 4 WRITE-UP Protocol Materials Lab coat and disposable gloves PCR reac on 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 tips Cup for discarded ps Micropipettor OpenPCR machine PCR Reaction Sample List Tube Label PCR Reaction Sample Patient ID G3 + Positive control none G3 - Negative control none G3 1-1 Patient 1, replicate 1 49652 G3 1-2 Patient 1, replicate 2 49652 G3 1-3 Patient 1, replicate 3 49652 G3 2-1 Patient 2, replicate 1 99085 G3 2-2 Patient 2, replicate 2 99085 G3 2-3 Patient 2, replicate 3 99085 DNA Sample Set-up Procedure Step 1: Get two patient ID numbers from a Teaching Assistant and have them written down.  Step 2: In the report labels will be placed on each tube to identify what specimen is in which tube. For Group 3, these tubes will be marked as G3 P, G3 N, G3 1-1, and etc. Step 3: The results will then be displayed in a graph which contains all specimen. 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 Q1. What is the function of each component of a PCR reaction? Template DNA: Provides optimal input amounts for PCR amplification Primers: Bind to 3' ends of the DNA where the desired template is Taq Polymerase: Replicates the target DNA Deoxyribonucleotides: Work as building blocks for the new DNA strands Q2. What happens to the components (listed above) during each step of thermal cycling? INITIAL STEP: 95°C for 2 minutes: The DNA separated due to the heat Denature at 95°C for 30 seconds: Strands are unpaired Anneal at 57°C for 30 seconds: Primers bind to the target DNA sequences Extend at 72°C for seconds: Taq DNA polymerase binds FINAL STEP: 72°C for 2 minutes: Begins adding nucleotides to the DNA FINAL HOLD: 4°C 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? Andenine (A) pairs with: Thymine(T) Thymine(T) pairs with: Andenine (A) Cytosine (C) pairs with: Guanine (G) Guanine (G) pairs with: Cytosine (C) Q4. During which two steps of thermal cycling does base‐pairing occur? Explain your answers. During the second step of thermal cycling the TAQ DNA polymerase begin to add nucleotides to the template DNA strands Part 1: Use the NCBI database to find a disease-associated sequence What is a nucleotide? small-scale multi-base deletions or insertions (also called deletion insertion polymorphisms or DIPs) What is a polymorphism? A common variation in the sequence of DNA among individuals. Genetic variations occurring in more than 1% of the population would be considered useful polymorphisms for genetic linkage analysis. What species is this variation found in? Homo sapiens What chromosome is the variation located on? 12:40315266 What is listed as the Clinical significance of this SNP? Uncertain significance What condition is linked to this SNP? Susceptibility to Parkinson’s disease Part 2 : Find the DNA sequence of the SNP, and the surrounding sequence What does LRRK2 stand for? Leucine rich repeat kinase 2 What is the function of LRRK2? Protein binding, Clathrin binding, MAP kinase kinase activity What is an 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. The disease-associated allele contains what codon? GAG What numerical position of the SNP? Position: 40315266 Part 3: Now that you have a good view of the DNA sequence, you can design primer for PCR Non-disease forward primer (20 nt)? 5'-TTAAGTGACTTGTACTTTGT-3' the numerical position exactly 20 bases to the right of the disease SNP is? 40315466 Non-disease forward primer (20 nt)? 5'-TGAAGCTCTTCAAGTAGTCT-3' Disease forward primer (20 nt)? 5'-TTAAGTGACTTGTACTTTGA-3' Disease reverse primer (20 nt)? 5'-TGAAGCTCTTCAAGTAGTCT-3' SNP Information & Primer Design Background: About the Disease SNP SNP stands for Single Nucleotide Polymorphisms, in which occurs in every 300 nucleotides on average. There are nearly 10 million SNPS in the human genome that plays a more direct role in disease by affecting the genes function. It has recently been proven that SNPS has help identify the response of the human body with certain drug uses. As well as tracing inheritance of disease within families. Overall, SNPS is a programmed bio-marker to the human use. Primer Design and Testing In the primer test the results are the following.In the nucleotide sequence, variations are used as positional markers. When placed in a unique location in a genome, the marker works with the same logic as a sequence STSs or framework microsatlellite markers. In other words the flake sequence services as a landmark in the genome. As shown in the figure below the flake base either assayed for variation. Error creating thumbnail: File missing