BME100 f2018:Group7 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: Daniella McFeely Role: Innovative Researcher Name: Jennifer Smetanick Role: Innovative Researcher Name: Ethan Armendariz Role: Innovative Researcher Name: Sebastian Tellez Role: Innovative Researcher Name: Fabiola Cervantes Role: Innovative Researcher LAB 4 WRITE-UP Protocol Materials Lab Coat Disposable gloves PCR reaction mix, 8 tubes, 50 μL each: Mix contains Taq DNA polymerase, MgCl, and dNTP's DNA/ primer mix, 8 tubes, 50 μL each: Each mix contains different template DNA. All tubes have the same forward primer and reverse primer A strip of empty PCR tubes Disposable pipette tips: use each only once Cup for discarded tips Micropipettor OpenPCR machine: shared by two groups PCR Reaction Sample List Tube Label PCR Reaction Sample Patient ID G7 + Positive control None G7 - Negative control None G7 1-1 Patient 1, replicate 1 28063 G7 1-2 Patient 1, replicate 2 28063 G7 1-3 Patient 1, replicate 3 28063 G7 2-1 Patient 2, replicate 1 67655 G7 2-2 Patient 2, replicate 2 67655 G7 2-3 Patient 2, replicate 3 67655 DNA Sample Set-up Procedure Step 1: Collect listed materials. Step 2: Divide the strip of empty PCR tubes in half to have two strips of four linked tubes. Step 3: Label side of empty tube in black marker with the Tube Labels described above. Step 4: Place the labeled tubes on the rack. Step 5: Use a new pipette to add 50 μL of the PCR reaction mix into positive control tube. Discard disposable tip. Step 6: Use a new pipette to add the 50 μL positive control DNA/primer mix to same tube. Step 7: Repeat steps 5-6 for the negative control, patient 1 (replicates 1-3) and patient 2 (replicates 1-3). The appropriate DNA/primer must be used. Step 8: Tightly close the lids on the PCR reaction tubes. Step 9: Place the PCR tubes in the thermal chamber. Start once all 16 slots are filled with other groups. OpenPCR Software Set-up Procedure Add New Experiment Insert Values below into the next OpenPCR interface 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 Extend at 72°C for 30 seconds FINAL STEP: 72°C for 2 minutes FINAL HOLD: 4°C PCR INTERFACE EXAMPLE: Run the test by clicking start Research and Development PCR - The Underlying Technology Original PCR Illustration Function of PCR Components Polymerase Chain Reaction (PCR) is a multistep process that utilizes multiple components, such as primers, template DNA, Taq DNA polymerase, and deoxyribonucleotides (dNTP's); each component has a specific function. Primers are short pieces of DNA that are made in a laboratory, and they attach to areas on the DNA strands at both of the ends of the segment you want to copy, with a low probability of identifying the wrong site. Taq DNA polymerase is an enzyme which preserves a genome during replication and is also a thermophile that can survive extremely high temperatures. Taq DNA polymerase is used in PCR due to the fact that DNA is heated to high temperatures during the PCR process; thus, an enzyme that is capable of withstanding high temperature is required. Deoxyribonucleotides are the four molecules (Adenine, Cytosine, Guanine, Thymine) that make up the DNA structure. The template DNA is the DNA that serves as the basis for replication during the PCR process. The Steps of Thermal Cycling At the initial step, when the DNA is heated to 95°C for 2 minutes, the DNA double helix unwinds, followed by the denaturing process, which creates two single strand DNA molecules. Upon completion of this step, the anneal process at 57°C for 30 seconds allows for single-stranded DNA molecules to attempt to pair up, but the primers in the tube lock their target before the strands can rejoin. During the extending process (at 72°C for 30 seconds), the temperature activates the DNA polymerase, which locates a primer attached to the DNA strand and begins to add the complementary nucleotides to the strand until it reaches the end. During the final process (a 72°C hold for 2 minutes) the two strands of the desired product begin to appear. Through the repetition of this process, the two strands will be multiplied into many. The final hold at 4°C is designed to maintain the PCR product until use. Nucleotide Pairing Nucleotide base pairs anneal to complimentary base pairs through a hydrogen bonding process. The base pairs anneal in a specific manner - Adenine will always pair with Thymine, and Guanine will always pair with Cytosine. Similarly, Thymine will always anneal with Adenine, and Cytosine with Guanine. Base Pairing during PCR process Nucleotide base pairs anneal to their complimentary counterparts during the extending process of thermal cycling, as well as the anneal process. The extending process allows for the DNA polymerase to produce the complimentary nucleotides to the strands of DNA, thus completing the sequence. SNP Information & Primer Design Background: About the Disease SNP Nucleotides are the building blocks (monomers) for nucleic acids. There are four types: Adenine (A), Thymine (T), Cytosine (C), and Guanine (G). A genetic polymorphism is a variance in the DNA. Thus, a SNP is a difference in a single nucleotide. This specific SNP being examined is found in Homo sapiens. It is located on chromosome 12:40315266. Clinical significance is unknown but according to PubMed, it is linked with familial parkinsonism. LRRK2 stands for leucine rich repeat kinase 2. The functions of LRRK2 include: ATP binding, GTP binding, and GTP-dependent protein kinase activity. An allele is a variant form of a gene located on a specific chromosome and at a specific position. The disease-associated allele contains the codon GAG. The SNP causes a change from Valine (GTC) to Glutamine (GAG). The numerical position of the SNP is 40315266. Primer Design and Testing The non-disease forward primer (20 nt) is 5'-TTAAGTGACTTGTACTTTGT-3' The non-disease reverse primer (20 nt) is 5'-TGAAGCTCTTCAAGTAGTCT-3' The disease forward primer (20 nt) is 5'-TTAAGTGACTTGTACTTTGA-3' The disease reverse primer (20 nt) is 5'-TGAAGCTCTTCAAGTAGTCT-3' The UCSC In-Silico PCR tool validated the results. The non-disease sequence was found in the human genome and the disease sequence was not because it is a mutation. Non-Disease Results Disease Results