BME100 s2017:Group4 W1030AM 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 Name: Chase Wallace Name: Kyle Xue Name: Lillie Robinson Name: Zoe Bargas) Brenna Hickey Name: Nicholas Holmes LAB 4 WRITE-UP Protocol Materials Lab coat and disposable gloves PCR reaction mix, 8 tubes, 50μL each: Mix contains Taq DNA polymerase, MgCl2, and dNTP’s DNA/ primer mix, 8 tubes, 50μL 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 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 50919 G4 1-2 Patient 1, replicate 2 50919 G4 1-3 Patient 1, replicate 3 50919 G4 2-1 Patient 2, replicate 1 67309 G4 2-2 Patient 2, replicate 2 67309 G4 2-3 Patient 2, replicate 3 67309 DNA Sample Set-up Procedure Label the empty PCR tubes with the appropriate descriptions. Two PCR tubes will be labeled as controls. The remaining 6 PCR tubes will be split into 2 sets of 3. One set will be the 3 replicates for patient 1 and will be labeled as such. The other set will be the 3 replicates for patient 2 and will be labeled as such. Place the empty PCR tubes onto a PCR tube rack to secure them in an upright position Use a micropipettor to move the pre-prepared 50μL PCR reaction mixes into their correspondingly labeled empty PCR tube. Make sure to use a new micropipettor tip each time. Use a micropipettor to move the pre-prepared 50μL DNA/Primer mixes into their correspondingly labeled PCR tube. Make sure to use a new micropipettor tip each time. Place the PCR reaction mix tubes in the thermocycler. OpenPCR program HEATED LID: 100°C INITIAL STEP: 95°C for 2 minutes (to allow the DNA to denature and separate as much as possible) NUMBER OF CYCLES: 25 Denature at 95°C for 30 seconds (to allow the DNA to uncoil and the strands to separate) Anneal at 57°C for 30 seconds (to allow primers to attach to the DNA at a certain spot) Extend at 72°C for 30 seconds (to allow DNA polymerase to extend the desired portion of the DNA strand) FINAL STEP: 72°C for 2 minutes (to ensure that the DNA has been fully elongated) FINAL HOLD: 4°C (to let the DNA, in its fully replicated state, coil back together and act like DNA naturally would as well as to hold further replication) Research and Development PCR - The Underlying Technology Q1: What is the function of each component of a PCR reaction? Template DNA The DNA strand that contains the desired region for replication. Primers They are used to target the region wanted for replication. Binds to DNA to initiate replication. Built to target that region only. Taq Polymerase It is used to synthesize and lengthen a new strand of DNA. Deoxyribonucleotides (dNTP’s) The building blocks of DNA. The four types (A,C,G,T) are added to make the new DNA strands during replication. Each base will attach themselves to their complementary base Q2: What happens to the components (listed above) during each step of thermal cycling? INITIAL STEP: 95 degrees Celsius for three minutes The template DNA heats up in this segment and begins to denature. All DNA strands are separated into single stranded DNA. Denature: at 95 degrees Celsius for 30 seconds DNA will unravel and the two strands of DNA come apart allowing for transcription to occur. Anneal: at 57 degrees Celsius for 30 seconds Primers anneal at this temperature at a specified area that will be replicated many times over the course of PCR. Extend: at 72 degrees Celsius for 30 seconds Polymerase is allowed to run the course of the DNA (based on where the primers annealed) and replicate DNA to form another double strand. FINAL STEP: at 72 degrees Celsius for three minutes This step ensures that as much DNA that can be replicated is replicated. Final Hold: at 4 degrees Celsius This hold keeps any further progress from happening. All components of PCR do not work at this temperature and the replicated target DNA strands remain intact. 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? If you need help, use the “Build a DNA Molecule” tool at http://learn.genetics.utah.edu/content/begin/dna/builddna/ Adenine (A): Thymine (T) Thymine (T): Adenine (A) Cytosine (C): Guanine (G) Guanine (G): Cytosine (C) Q4: During which two steps of thermal cycling does base-pairing occur? Explain your answers Base-pairing occurs during annealing and elongation. During annealing, the primers will anneal to the target DNA; they have been designed to bind to a specific region of the DNA. Its bases will pair with the bases in the target DNA segment. During elongation, TAQ Polymerase will extend the primers by adding dNTPs to the target strand and make double stranded DNA. Extra Credit PCR Original Illustration At around 95 degrees Celsius, the hydrogen bonds between the AT and GC pairings are broken, which allows the template DNA to unzip and separate. At around 57 degrees Celsius, 2 primers anneal to the desired region in the template DNA. They attach to the 3' ends of the top and bottom strand because DNA polymerase always builds 5' to 3'. At around 72 degrees Celsius, the DNA polymerase becomes active and attaches the complementary nucleotide bases until it reaches the end. Taq polymerase is used in particular because it operates well at high temperatures. After elongation occurs, the whole denaturing, annealing, and elongating process repeats itself for around 25 cycles in this case. After the third cycle, DNA segments that are purely the desired regions begin to appear. After all the cycles, millions to over a billion copies(depending on the number of cycles) of the desired DNA region will have been replicated. SNP Information & Primer Design Background: About the Disease SNP A SNP is a single nucleotide polymorphism, which means that one nucleotide is replaced with another(the standard nucleotide is replaced with a different one due to a point mutation). In the specific case of this disease SNP, an adenine nucleotide is replaced with a cytosine instead. The allele changes from AGT to CGT when this SNP occurs. Although the majority of SNPs do not have negative disease related consequences, this specific SNP is pathogenic. In particular, it is linked with Cystic Fibrosis and is found on chromosome 7:117587799 in Homo sapiens(humans). General Background Information: What is a nucleotide? A nucleotide is a compound consisting of a nucleoside linked to a phosphate group. The nucleotides form the basic structural unit of nucleic acids such as DNA. What is a polymorphism? A polymorphism is the condition of multiple forms of a single gene. Primer Design and Testing Our goal was to find the allele responsible for the CTFR gene, which is associated with cystic fibrosis. The allele is supposed to contain “AGT”, however, the disease-associated allele contains the bases CGT, becoming arginine. We located this allele, and found it in the human genome, to better understand the process of genetic modification. There was no result for the disease positive allele. This is because it is a mutation, so it is not part of the normal human genome. While this mutation occurs, it does not happen to the average human, so it was unable to be found in the database. This screenshot shows that we have found the correct non disease forward primer. This is important because we need both the correct forward and reverse primers in order to adjust the genetic code. With these primers, we now have the chromosome and location on the chromosome that this gene is located.