8 tubes DNA/ primer mix (50 µL each). Each mix: different template DNA, but same forward and reverse primers
Equipment
Strip of empty PCR tubes
Disposable pipette tips (single use only use)
Cup for discarded tips
Micropipettor
OpenPCR machine
PCR Reaction Sample List
Tube Label
PCR Reaction Sample
Patient ID
G2 +
Positive control
none
G2 -
Negative control
none
G2 1-1
Patient 1, replicate 1
95353
G2 1-2
Patient 1, replicate 2
95353
G2 1-3
Patient 1, replicate 3
95353
G2 2-1
Patient 2, replicate 1
64816
G2 2-2
Patient 2, replicate 2
64816
G2 2-3
Patient 2, replicate 3
64816
DNA Sample Set-up Procedure
Procedure:
1. Extract the DNA from the source
2. Put the extracted DNA into the test tube
3. Add primer 1 to the PCR tube. Primers attach to sites on the DNA strands that are at either end of the segment you want to copy.
4. Add primer 2 to the PCR test tube
5. Add nucleotides to the PCR tube
6. Add the DNA polymerase to the PCR tube
7. Place the PCR tube into a DNA Thermal Cycler
8. Allow the thermocycler to complete 30 cycles
9. Place in refrigerator until later use
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
Research and Development
Q1. What is the function of each component of a PCR reaction?
Template DNA:
The non-coding strand in DNA that is copied, this strand is used for genetic information. The template strand soon allows the protein to be complementary and parallel.
Primers:
Short pieces of DNA that have any sequence of nucleotides desired and match the segment of DNA you want copied. One primer attaches to the top strand at one end, and the other primer attaches to the bottom strand at the other end.
Taq Polymerase:
A naturally occurring complex of proteins whose function is to copy a cell’s DNA before division. The DNA polymerase attaches itself near the end of the primer and starts adding nucleotides.
Deoxyribonucleotides (dNTP’s):
Building blocks for new DNA strands. Consists of A’s, C’s, T’s, and G’s
Q2. What happens to the components (listed above) during each step of thermal cycling?
INITIAL STEP: 95°C for 3 minutes:
When there is a template that is rich in C-G sequences, this longer process is required fully denature the template.
Denature at 95°C for 30 seconds:
Template DNA double helix separates, creating two single-stranded DNA molecules.
Anneal at 57°C for 30 seconds:
Primers lock onto target single strands before they can rejoin.
.
Extend at 72°C for 30 seconds:
DNA polymerase activates and locates primer attached to single-strand DNA. It continually adds complementary nucleotides onto strand until it reaches the end and falls off.
FINAL STEP: 72°C for 3 minutes:
The desired products appear. These are the DNA copies of the segment of DNA targeted. After 30 cycles, over a billion fragments that contain the target sequence are present.
FINAL HOLD: 4°C:
In order to maintain the products of the PCR in which there is time to transfer the product to a more suitable environment until the products can be used for further analysis and experiment.
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.
Base pairing first occurs in the third step of the process where the primers attach to the single-stranded DNA. The primers have a certain sequence of nucleotides that are base-paired to a certain section of the DNA, which is how the primer knows where to attach. Then, in the following step, base pairing occurs again as DNA polymerase grabs free-floating nucleotides and adds them along the single-strand of DNA, matching A and T together and G and C together.
PCR - The Underlying Technology
PCR, which stands for polymerase chain reaction, is a biochemical technique that uses enzymes and thermal cycling to copy DNA strands. The main components to PCR are polymerase, template DNA, primers, nucleotides, and buffer. The polymerases are enzymes that assemble new strands of DNA from templates and nucleotides. Taq polymerase is especially formulated to withstand the high temperature associated with PCR reactions. Another important component is template DNA. This template DNA is the DNA that will be read and copied by the polymerase. The template strand allows for the proteins to be complementary and parallel. Another component are the primers. Primers are short fragments of synthesized DNA that bind to the template. There is a forward primer which starts the PCR and a reverse primer which designates the end of the PCR. Another component of PCR are the nucleotides. These nucleotides are necessary for making DNA copies. Deoxynucleoside triphosphates (dNTPSs) will be used in the PCR. These are the primary components of PCR.
Then, these components undergo a process of thermocycling where the DNA is initialized, denatured, annealed, extended, and elongated. In the process of initialization, the solution is heated at 95 degrees Celsius where in order to prepare the solution for the denaturation. This step is especially important for DNA sequences that are rich in C-G content and when hot-start polymerases need to be activated. The next step is denaturation. In this step, the reaction is heated to 95 degrees C in which it breaks down the DNA and primers which allow them to anneal in the next step. Following denaturation, the annealing step allows the formation of the double-stranded DNA structures by having the polymerase bind to the primer/taq DNA. In the extension step, the polymerase reads and copies the template DNA thus creating new double-stranded pieces of DNA. In the final elongation, te polymerases finish reading their current strand. This step helps to reduce the number of truncated copies in the final product.
SNP Information & Primer Design
Background: About the Disease SNP
Single Nucleotide Polymorphisms (SNPs) are changes in your basic building block: nucleotide. These slight genetic variations are very frequent and one can have roughly million SNPs in their genome. Although most don't cause any health or development issues, some can be very dangerous
Primer Design and Testing
First, the the DNA sequence of the SNP associated with the disease was identified. The first 20 bases pairs were used to identify the forward primer, then 200 bases pairs away we used the last 20 base pairs for the reverse primer. Then, for the disease coding primers, we altered the base pair in a codon "CTC" to "ATC," which is the mutation that causes this disease. With that alteration, we were able to produce forward and reverse primers for the disease-associated allele.
BME 100 Fall 2016
