Mix contains Taq DNA polymerase, MgCl2, 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
Cup for discarded tips
Micropipettor
OpenPCR machine
PCR Reaction Sample List
Tube Label
PCR Reaction Sample
Patient ID
G3 +
Positive control
none
G3 -
Negative control
none
G3 1-1
Patient 1, replicate 1
14149
G3 1-2
Patient 1, replicate 2
14149
G3 1-3
Patient 1, replicate 3
14149
G3 2-1
Patient 2, replicate 1
42390
G3 2-2
Patient 2, replicate 2
42390
G3 2-3
Patient 2, replicate 3
42390
DNA Sample Set-up Procedure
Collect materials and put them on ice within a container.
Label each PCR tube as followed: 1 tube as negative, 1 as positive, 3 as patient 1, and 3 as patient 2.
Add 50 μL PCR reaction mix to each PCR tubes.
Exchange the pipette for a new one between uses.
Add patient 1’s DNA/primer of 50 μL to the 3 PCR tubes labelled accordingly. Repeat this step for patient 2.
Add the negative control group’s DNA/primer 50 μL to the 1 PCR tube labelled accordingly. Repeat this step for the positive control group.
Place the PCR tubes into the thermal cylinder.
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
A thermal cycler is used to enhance portions of DNA by way of the polymerase chain reaction (PCR). This is done by repeatedly heating and cooling to force temperature dependant reactions. As it cools the DNA separates and the primers attach. Then as it heats up the primer completes the DNA that it is attached to. This fluctuation of temperature continues in order to multiply the DNA strands.
Research and Development
PCR - The Underlying Technology
Q1. What is the function of each component of a PCR Reaction?
Template DNA is the DNA to be copied in the PCR reaction.
Primers attach to each end of the targeted segment wanted to copy. The second primer attaches to the second site.
Taq Polymerase read the DNA and matches nucleotides in order to create copies.
Deoxyribonucleotides (dNTP’s) are single pieces of DNA known as A, T, G, and C which are needed in order to copy the original base pairs.
Q2. What happens to the components (listed above) during each step of thermal cycling?
The initial step is to raise the temperature to 95°C for 3 minutes. The hydrogen bonds weaken for the separation in the denature. The second step is denature at 95°C for 30 seconds. This causes the template DNA to separate making two separate strands of DNA. Next is anneal at 57°C so the primers will begin to attach to the strands of Template DNA. The next step is to extend at 72°C for 30 seconds. The DNA polymerase activates and creates complementary base pairs of deoxyribonucleotides to replicate the DNA and will continue to replicate until it falls off the end of the strand. The final step of 72°C for 3 minutes allows the polymerase to finish reading the DNA strands,finalizing the replication. Finally, with a hold at 4°C, the PCR process is complete and the new strands of targeted DNA are ready to be transferred into a more permanent storage.
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?
Adenine (A): T
Thymine (T): A
Cytosine (C): G
Guanine (G): C
Q4. During which two steps of thermal cycling does base-pairing occur? Explain your answers.
The base-pairing begins to occur during the extend at 72°C for 30 seconds after the primer has already been engaged and the DNA polymerase begins to create complementary base pairs of deoxyribonucleotides and this will continue until the the DNA polymerase reaches the end of the strand and falls off.
EXTRA CREDIT
SNP Information & Primer Design
Background: About the Disease SNP
An SNP is a single nucleotide polymorphism, meaning that a change in only a single DNA base pair leads to a mutation. SNPs, which occur throughout a person’s DNA, are the most common form of genetic variation in people. Most of the time, SNPs do not have a negative effect on people’s health. However, some SNPs are responsible for the inheritance of diseases within families. The specific SNP that we are analyzing is the replacement of the codon AGT with the codon CGT (the single base Adenine is swapped for Cytosine). This occurs in the 7th chromosome. The SNP causes cystic fibrosis, which causes a buildup of mucys in the lungs, pancreas, and other organs.
Primer Design and Testing
The non-disease forward and reverse primers were found by analyzing the gene with the SNP. The mutated nucleotide (SNP) was found, and the 20 nucleotides leading up to this became the forward primer. The reverse primer was similarly found using the bottom strand of DNA, including 20 nucleotides from a position 200 bases away from the SNP. The diseased forward primer is identical to the non-disease primer, except the final A nucleotide is a C nucleotide, indicating the mutation. The disease reverse primer is identical to the non-disease reverse primer. These primers were tested and found to be correct through a given human genome database.
BME 100 Spring 2017
