Name: Derek Scott
Name: Parker Storry
Name: Alexander Dasilva
Name: Luis Morales
Name: Ksenia Poujlivaia
Name: Shivanshi Shukla
LAB 4 WRITE-UP
- Lab coat
- Disposable gloves
- PCR reaction mix
- 8 Tubes, 50 μL: Mix contains Taq DNA polymerase, MgCl2, and dNTP’s (http://www.promega.com/resources/protocols/product-information-sheets/g/gotaq-colorless-master-mix-m714-protocol/)
- 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 re-use disposable pipette tips or samples will be cross-contaminated
- Cup for discarded tips
- OpenPCR machine: shared by two groups
PCR Reaction Sample List
| Tube Label || PCR Reaction Sample || Patient ID
| G13 + || Positive control || none
| G13 - || Negative control || none
| G13 1-1 || Patient 1, replicate 1 || 24231
| G13 1-2 || Patient 1, replicate 2 || 24231
| G13 1-3 || Patient 1, replicate 3 || 24231
| G13 2-1 || Patient 2, replicate 1 || 48862
| G13 2-2 || Patient 2, replicate 2 || 48862
| G13 2-3 || Patient 2, replicate 3 || 48862
DNA Sample Set-up Procedure
- Gather and assemble all materials
- Label all tubes and organize PCR reaction mix with DNA primer list
- Create two strips of four linked, empty PCR tubes by cutting the line of tubes
- Label the side of the PCR tubes
- Place PCR tubes in a rack
- Place a new pipette tip onto the micropipettor
- Extract 50 μL of PCR reaction mix into pipette tip and place it into the corresponding PCR tube
- Discard pipette tip into disposal, and insert a new pipette tip
- Extract 50 μL DNA/ primer mix into the pipette tip
- Dispense the DNA/primer mix used in previous step and discard pipette tip in disposal
- Close PCR tube lid tightly
- Repeat steps 6-11 to complete filling the remaining PCR tubes
- Place finished PCR tubes into PCR machine
Research and Development
PCR - The Underlying Technology
What is the function of each component of a PCR reaction?
The template DNA used in a PCR reaction is the piece of information that contains the desired DNA sequence to be multiplied throughout the process. In the PCR, the template DNA is heated to 95 Celsius, causing a denaturing, and creating a separation between the two strands. Once separated, the temperature is quickly dropped to 57 Celsius to cause the primer, a single strand of DNA complementary to the primary target sequence that allows the taq DNA polymerase to attach, to anneal and attach to the template DNA. The temperature is then raised to 72 Celsius, the Taq DNA polymerase attaches to the DNA sequence at the primer, and attaches deoxynucleotides, single A, G, C, or T bases, to synthesize the DNA copy through extension.
Through the temperature cycling, this process is repeated multiple times and the DNA target sequence is amplified exponentially.
What happens to the components (listed above) during each step of thermal cycling?
The initial step is to allow the template DNA to break down by heating it to 95 Celsius for three minutes. During subsequent denaturing, the time at such a high temperature is shortened to not limit the efficacy of taq DNA polymerase, or other components. During denaturing, the template DNA strand is broken into individual strands due to the breaking of the hydrogen bonds between the base pairs by heating the mixture to 95 Celsius for 30 second. At this time, the taq DNA polymerase, deoxynucleotides, and primers are in the mixture but not interacting. After the temperature is decreased and PCR enters the process of annealing and reaches 57 Celsius for 30 second, the buffer attaches itself to complementary base pair sequences on both template DNA strands.
As the temperature is further increased to 72 Celsius for 30 seconds, the taq DNA polymerase attaches itself at the primer, and synthesizes a copied strand of DNA from the template by assembling the base pairs using the deoxynucleotides. This causes the new strand of DNA to extend. This process is repeated multiple times to create exponential growth of the target DNA sequence.
In the final step of the process, the mixture is held to 72 Celsius for 3 minutes to allow the taq polymerase ample time to completely finish any sequence copying it is currently processing. The final hold is at 4 Celsius to keep the DNA viable until it is used.
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?
The base adenine pairs with the base thymine. They share two hydrogen bonds between the molecules. The pair cytosine pairs with guanine and contains three hydrogen bonds between molecules.
During which two steps of thermal cycling does base-pairing occur? Explain your answers.
Base-pairing occurs during the extension phase. During this phase, taq DNA polymerase has attached to the primer, only attached in the previous annealing stage, and uses the deoxynucleotides floating in the mixture to extend the DNA sequence. During earlier phases, the template DNA had no primer and the temperature was too high for a primer to be added (denaturing), or the primer had just attached, but the taq DNA polymerase had not attached to the primer (Annealing). In phases after extension, the mixture is heated again causing the denaturing of the bonds holding the DNA replicated sequence to the template DNA.
SNP Information & Primer Design
Background: About the Disease SNP
The disease single nucleotide polymorphism, or SNP, is the allele on the DNA strand at a certain point that is recognized as the cause of a certain disease. In this case, the SNP is a pathogen associated with the MC1R gene. The MC1R gene (Melanocortin 1 Receptor) gives instructions for the creation of a protein that plays a significant role in pigmentation. However, this gene leads to an increased risk of skin cancer. The gene's location is on the 16th chromosome at 16:89919736.
Primer Design and Testing
The primer is designed by first locating the allele associated with the disease of interest on the DNA strand. Once located and its position has been noted, the 20 base forward primer can be derived. In this case, the non-disease forward primer is: CAGCATCGTGACCCTGCCGC.
To find the reverse primer, the position 200 bases to the right of the SNP is found. From there, the first 20 bases from the bottom strand of DNA are recorded from right to left, resulting in a non-disease reverse primer of: CTTGTGGAGCCGGGCGATGC.
The disease forward primer is derived by replacing the last nucleotide of the non-diseased forward primer with the base nucleotide from the SNP allele. This replaces a C with a T, resulting in a disease forward primer of: CAGCATCGTGACCCTGCCGT.
The disease reverse primer is the same as the non-diseased reverse primer.
After testing the primers with the UCSC PCR testing website, it was determined that the non-diseased primers would work. The results were 220 bp, the length of the working sequence. The non-diseased primers, on the other hand, did not have any matches. This is because the primers are sequences of DNA that match the human genome, except for the final nucleotide in the forward primer, which is the SNP. In the diseased forward primer, this difference from the nucleotide from the human genome explaining which explains the lack of matches.