BME100 f2018:Group6 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 Name: Zachery Shropshire Name: Siris Smith Name: Adriana Gillespie Name: Anna Haworth Role(s): Research and Development Section Name: Sarah Ruby LAB 4 WRITE-UP Protocol Group 6 Materials Standardized PPE for lab Start with a PCR reaction mix, also 8 tubes, 50 μL each: Your mix will contain a Taq DNA polymerase, MgCl2, and dNTP’s You will also Need a DNA/ primer mix, also 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, the samples will become cross-contaminated A Cup for discarded tips A Micropipettor OpenPCR machine: shared by two groups Group 6 PCR Reaction Sample List Tube Label PCR Reaction Sample Patient ID G6 + Positive control none G6 - Negative control none G6 1-1 Patient 1, replicate 1 23643 G6 1-2 Patient 1, replicate 2 23643 G6 1-3 Patient 1, replicate 3 23643 G6 2-1 Patient 2, replicate 1 47311 G6 2-2 Patient 2, replicate 2 47311 G6 2-3 Patient 2, replicate 3 47311 DNA Sample Set-up Procedure Have everyone working in the lab put on proper PPE. Collect the necessary materials. Be sure to never reuse a pipette tip--discard after every use into the designated cup. Make sure the PCR tubes are properly labeled with the PCR reaction sample contained inside. Add one tube of the primer mix into the positive control PCR tube. Add one tube of the PCR reaction mix into the positive control PCR tube. Repeat steps 2 and 3 for the negative control and every patient sample. Place the tubes into the thermal cycler. PCR Steps The first step will be heating the temperature to 95°C for 2 minutes. There will be a total of 25 cycles: The temperature will first be set to 95°C so the DNA will denature. The temperature will then decrease to 57°C, which will allow the DNA primers to attach to the template strand. This process is also known as annealing. Finally, the temperature will rise to 72°C to allow the Taq DNA polymerase enzyme to synthesize the new strand. Each of these steps will take 30 seconds. The final step has the temperature at 72°C for 2 minutes After the completion of PCR, the DNA will be stored at 4°C to preserve the sample. Research and Development PCR - The Underlying Technology Introduction PCR, or Polymerase Chain Reaction, is commonly used to amplify a certain DNA sequence for purposes of studying the gene or testing for diseases. Our group is using PCR to determine whether our two patient samples carry the single-nucleotide polymorphism disease (SNP) by amplifying the gene segment where the SNP is located. The non-diseased sequence is usually Valine (GTG) but in SNP, it is altered to Glutamine (GAG). PCR Components Amplifying the gene segment using PCR requires a Template DNA, 2 Primers, DNA Taq Polymerase, Deoxyribonucleotides (dNTP’s) and an OpenPCR machine. The Template DNA will have the gene segment needed to be copied. The Primers, one forward and one reverse, give a place for Taq polymerase to start coding and attach at the start of the segment that is being copied. Taq polymerase is an enzyme that comes from Thermus aquaticus and can replicate DNA at high temperatures. The dNTP’s are the source of nucleotides for building DNA that Taq polymerase will use to build the new strand. The OpenPCR machine is a Thermal cycling unit that allows the PCR reaction to occur. The PCR reaction in the thermocycler process is the following: Heat up the thermocycler to 95°C (2 minutes) The DNA will denature at 95°C (30 seconds) Primers will bind at 57°C (30 seconds) Taq polymerase will create the new strand of DNA at 72°C (30 seconds) Repeat steps 2-5 25 times giving the process of denaturing, annealing, and extending Hold DNA in cycler for 2 minutes at 72°C Refrigerate DNA samples at 4°C to preserve them The DNA Our replicated DNA will have millions of copies of itself after PCR, but how did it make the DNA? The DNA was made using the Nucleotides brought by the dNTPs. The dNTP contains the nucleotides Adenine (A), Thymine (T), Cytosine (C), and Guanine (G). These 4 nucleotides make up all DNA by pairing themselves together (A to T and G to C) by hydrogen bonding which stick them together. This occurs in the Extend part of the cycle and finishes in the final step in the Thermal cycler. SNP Information & Primer Design Background: About the Disease SNP "SNP" stands for single-nucleotide polymorphism. The nucleotide is a building block that makes up DNA. In all its four different types of DNA nucleotides, with the job of connecting the two halves of a DNA double helix; adenine will always attach to the thymine, and the guanine will always attach to cytosine. The polymorphism varies in common ways of separate DNA strands. They're very rare, happening in just over 1% of the population and it’s useful for genetic analysis. The SNP code at rs721710 will be used for this lab, and the chromosome variation should be located at 12:40315266. The clinical significance will be uncertain, linking the SNP to Parkinson's disease. The DNA Sequence found should be LRRK2, which stands for leucine-rich repeat kinase 2, consisting of ATP binding, GTP binding, GTP-dependent protein kinase activity in the body. The numerical position of the SNP is located at 40315266. Primer Design and Testing To design a non-disease forward primer, the sequence that was 20 bases long (20 nt) ending in a nucleotide at 40315266 was recorded. Non-disease forward primer (20 nt): 5’- T T A A G T G A C T T G T A C T T T G T. The oligonucleotide sequences were always written with 5’ end at the left of the sequence and a 3’ on the right end of the sequence. Every PCR reaction needs two primers to amplify the DNA. To design a non-disease reverse primer, go 200 bases to the right of the 40315266 nucleotide. The reverse primer is read from the bottom strand of DNA nucleotides. It is important to be very careful in how this strand is read and written, as the bottom strand must be read from right to left this way, and written left to right. The non-disease reverse primer was read as (20 nt) 5’- T G A A G C T C T T C A A G T A G T C T. The group then designed two disease SNP-specific primers, simply by changing the last base of the non-disease forward primer so that it reads as the disease SNP nucleotide. The disease forward primer was (20 nt) 5’- T T A A G T G A C T T G T A C T T T G A. The disease reverse primer was the same as the non-disease reverse primer: (20 nt): 5’- T G A A G C T C T T C A A G T A G T C T. Last but not least, our group validated the primers using a non-disease human genome sequence. Plugging in all of the recorded data given with the settings advised, the non-disease primers showed up as a valid sequence, while the disease forward primers did not. This screengrab shows the result from the non-disease primers; the sequence exists. This screengrab shows the result from the disease primers.