BME100 s2017:Group4 W8AM L4

BME 100 Spring 2017	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 Bat-El Shabtai Katie Masterson Tim Styrlund Mustafa Alsadiq Sydney Horton LAB 4 WRITE-UP Protocol Materials Lab coat and disposable gloves PCR reaction mix, 8 tubes, 50 μL each: Mix contains Taq DNA polymerase, MgCl 2, 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: only use each only once. Never reuse disposable pipette 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 G4 + Positive control none G4 - Negative control none G4 1-1 Patient 1, replicate 1 92884 G4 1-2 Patient 1, replicate 2 92884 G4 1-3 Patient 1, replicate 3 92884 G4 2-1 Patient 2, replicate 1 70752 G4 2-2 Patient 2, replicate 2 70752 G4 2-3 Patient 2, replicate 3 70752 DNA Sample Set-up Procedure Cut the strip of of empty PCR tubes in half to create four linked tubes. Label the sides of the tubes. Place all four tubes in a rack. Transfer 50 microliters of PCR reaction mix into the empty tube labeled positive control and discard the tip. Using a new pipette tip, transfer 50 microliters of the positive control DNA/ primer mix into the positive control PCR tube. This tube should now contain a total of 100 microliters. Repeat steps 4 and 5 an additional 7 times: for the negative control, for each 3 replicates of patient 1, and each 3 replicates of patient 2. For each tube make sure the corresponding DNA/primer mix is added. Close the lids of the PCR tubes tightly. Transfer all the tubes to the PCR machine, placing each tube in the slots in the heating block until all 16 slots are filled. Start the PCR machine. OpenPCR program HEATED LID: 100°C INITIAL STEP: 95°C NUMBER OF CYCLES: 25 (Denature at 95°C for 30 seconds, Anneal at 57°C for 30 seconds and Extend for at 72°C for 30 seconds) FINAL STEP: 72°C for 2 minutes FINAL HOLD: 4°C Research and Development PCR - The Underlying Technology What is the function of each component of a PCR reaction? The Template DNA is used as a guide to make the replicate DNA. Primers​ attach to sites on the DNA strands that are at either end of the segment that is being copied. The Taq DNA Polymerase reads the DNA code and attaches matching nucleotides to create DNA copies. The deoxyribonucleotides (dNTP’s) are genetic building blocks that are used to create the replicate DNA strands. What happens to the components (listed above) during each step of thermal cycling? ​During the initial step at 95°C for 3 minutes​, the DNA double helix separates, creating two single-stranded DNA molecules. During the denature​ step at 95°C for 30 seconds​, the DNA double helix separates, creating two single-stranded DNA molecules. During annealing at 57°C for 30 seconds​, the primer sequences bind to specific areas on the single-stranded DNA. During it's extension at 72°C for 30 seconds​, the DNA polymerase finds the primer and attaches to the DNA strand and begins to add complementary nucleotides onto the strand. During the final step at 72°C for 3 minutes, the DNA polymerase finishes adding to the DNA strand and falls off. During the final hold at 4°C, the DNA stand cools down to ensure that everything has recombined fully. 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? there are four nucleotides that are the basis of the DNA molecule. The four nucleotides are adenine, thymine, cytosine, and guanine. Due to the number of available spaces for hydrogen bonding, adenine binds with thymine and cytosine binds with guanine. During which two steps of thermal cycling does base-pairing occur? Base-pairing occurs during annealing and extension. In annealing, the nucleotides of the primers bind with their complementary base pairs on the DNA strands. In extension, DNA polymerase attaches complementary base pairs to the base nucleotides on the DNA strand. Illustration In order for the primers to be able to bind with the cancer DNA template, the template DNA must first be denatured which produces two complementary single-stranded DNA molecules. Once the DNA is denatured and the temperature is lowered to the optimum temperature, the primers pair up with specific sites on the template DNA where the nucleotides of the DNA are perfectly complementary to the nucleotides of the primer. The Taq polymerase then binds to the primers that have attached to the sites on the template DNA in preparation for amplifying the DNA. The Taq polymerase begins moving along the template DNA, attaching complementary nucleotides to the DNA as it progresses. Once the Taq polymerases have finished the template DNA strand, they detach and the DNA has been amplified. (Pictures taken from http://learn.genetics.utah.edu/content/labs/pcr/) SNP Information & Primer Design Background: About the Disease SNP SNP stands for "single nucleotide polymorphism." A nucleotide is an organic molecules that serve as the monomer units for forming the nucleic acid polymers DNA and a polymorphism is 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. When searching our specific SNP, it was found that the condition linked to this one was cystic fibrosis. This is found in homo sapiens and is located on the chromosome 7:117587799. Under the GeneView of this disease, we found out that CTFR stands for cystic fibrosis transmembrane conductance regulator. The non-disease allele associated with this is AGT, and the disease-associated allele is CGT. Primer Design and Testing The numerical position of the SNP is 117587799. The non-disease forward primer was found to be 5’- A G A A G G T G G A A T C A C A C T G A, while the non-disease reverse primer was found to be 5’- C A T T A T T T A T A G T T C T T A A A. The disease forward primer was found to be 5’- ​A G A A G G T G G A A T C A C A C C G T and the disease reverse primer was 5’- C A T T A T T T A T A G T T C T T A A T. When ran through the In-Silico PCR website, the non-disease primer resulted in 220bp sequence from the chromosome indicating the primer worked because it is a known sequence. The disease- specific primer designed resulted in no matches, because of a mutation in the DNA sequence, which matches what was expected of the diseased primer. Non-disease primer results: Disease primer results: Sources "PCR Virtual Lab" http://learn.genetics.utah.edu/content/labs/pcr/