BME100 f2018:Group12 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 Kami Leka Role(s) Omar Rivera Role(s) Michelle Sorsher Role(s) Cassidy Michaels Role(s) Ian Salgado Role(s) LAB 4 WRITE-UP Protocol Materials Lab coat and disposable gloves PCR reaction mix, 8 tubes, 50 microliters each: Mix contains Taq DNA polymerase, MgCl2, and dNTP's DNA/primer mix, 8 tubes, 50 microliters 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 pippette tips. If you do, the samples will become cross-contaminated. Cup for discarded tips Micropipettor OpenPCR machine: shared by two groups PCR Reaction Sample List Tube Label PCR Reaction Sample Patient ID G12 + Positive control none G12 - Negative control none G12 1-1 Patient 1, replicate 1 69578 G12 1-2 Patient 1, replicate 2 69578 G12 1-3 Patient 1, replicate 3 69578 G12 2-1 Patient 2, replicate 1 58152 G12 2-2 Patient 2, replicate 2 58152 G12 2-3 Patient 2, replicate 3 58152 DNA Sample Set-up Procedure 1. The Extracted DNA should be placed into a special PCR tube using a micropipette. 2. Add Primer 1 to the same PCR tube using a micropipette. 3. Add Primer 2 to the PCR tube using a micropipette. 4. Add the nucleotides to the PCR tube using a micropipette. 5. Add the DNA Polymerase to the PCR tube using a micropipette. 6. Place the filled PCR tube into the DNA thermal cycler. 7. Let the thermal cycler run until the desired amount of fragments that contain only the targeted sequence are made. 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 The function of each component of the PCR reaction: The template DNA is the DNA strand that is transcribed by the DNA polymerase. The primers are short pieces of laboratory-made DNA. They attach themselves to sites on the DNA strands on either end of the segment that is being copied. The segment is copied by Taq DNA polymerase which adds complementary nucleotides, also known as the Deoxyribonucleotides (dNTP’s), to the new strand of DNA. The steps of thermal cycling During the first step, the chamber is at 95 degrees for two minutes. During this stage the original DNA molecule splits into two strands creating single stranded molecules. For each cycle after this, the denaturation stage, where the DNA strands separate, will only last 30 seconds. Next, the temperature decreases to 57 degrees Celsius for 30 seconds and allows the primers to attach to their proper spots on the DNA strands. Then, the temperature increases to 72 degrees Celsius for 30 seconds to allow the Taq DNA polymerase to attach to the primers and adds the complementary nucleotides to the new strand. The DNA polymerase falls off when it reaches the end of the strand denoted by the second primer. This cycle will repeat until many copies of the gene have been made. Then the temperature will drop to 4 degrees Celsius where the DNA molecules are stable and double stranded. Base Pairing: Each nucleotide has a complementary nucleotide that it pairs up with. Adenine (A) pairs up with Thymine (T) and vice versa; Cytosine (C) pairs up with Guanine (G) and vice versa. They pair up this way as each pair is the same width as the purines and pyrimidines are different widths so one purine must pair up with one pyrimidine. The specific purine to pyrimidine pair stems from the specific locations of the hydrogen bonds on each molecule. When does base pairing occur: Base pairing occurs during the extend step at 72 degrees C because this is where the Taq DNa polymerase attaches to the primer and adds the complementary dNTP’s to the strand by pairing them by using base-pairing. Base pairing also occurs during the anneal step where the primer attaches to the DNA strand. The primer uses base pairing to connect to the correct location on the DNA strand. SNP Information & Primer Design Background: About the Disease SNP A nucleotide is a compound consisting of a nucleoside linked to a phosphate group. Nucleotides form the basic structural unit of nucleic acids such as DNA. They also form the base pairs in DNA that determine the alleles of different genes and later determine the amino acids used to create the proteins. Polymorphism is the presence of genetic variation within a population, upon which natural selection can operate. The species upon which the variation found is Homo Sapiens; the location of the chromosome was located on 12:40315266. The clinical significance of this SNP is uncertain but it is linked to familial parkinsonism. LRRK2 stands for Leucine rich repeat kinase 2. It serves as an integral part of ATP binding, GTP binding, and GTP-dependent protein kinase activity as well as many other functions in the cell. 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 healthy allele contains the codon GAG and the unhealthy allele contains the codon GAG and the numerical position for the SNP is 40315266. Primer Design and Testing We designed disease and non-disease primers using the human genome sequence. Each primer is 20 base pairs long. When the non-disease primer was tested using the UCSC In-Silico PCR website the result was a 220 base pair long sequence. When the disease primer was tested using the same website there were no matches.