PCR reaction mix, 8 tubes, 50 μL each: 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. Never reuse disposable pipette tips.
Cup for discarded tips
Open PCR Machine
PCR Reaction Sample List
Tube Label
PCR Reaction Sample
Patient ID
G11 +
Positive control
none
G11 -
Negative control
none
G11 1-1
Patient 1, replicate 1
75247
G11 1-2
Patient 1, replicate 2
75247
G11 1-3
Patient 1, replicate 3
75247
G11 2-1
Patient 2, replicate 1
55425
G11 2-2
Patient 2, replicate 2
55425
G11 2-3
Patient 2, replicate 3
55425
DNA Sample Set-up Procedure Step 1: Determine the Sample replicate compared to the table Step 2: Write The Patient number and the replicate number on the tube (ex.) 1-1 Step 3:Fill the PCR tube with the extracted DNA Step 4:Add Primer one to the PCR tube Step 5:Add Primer two to the PCR tube Step 6:Add nucleotides to the PCR tube Step 7:Add the DNA polymerase to the PCR tube Step 8:Place tube into the thermal cycler
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
PCR - The Underlying Technology
Function of PCR Reaction Components
In a PCR reaction, the template DNA is the extracted DNA that is going to be copied. Primers are short DNA sequences that bind to separated strands of DNA to copy DNA sequences. Taq polymerase is the protein that adds nucleotides to extend the strand where a primer is attached to a single DNA strand. Deoxyribonucleotides (dNTP's) are the components in the DNA that are being copied. There are four bases: adenine (A), guanine(G), cytosine(C), and thymine(T).
Thermal Cycling
The inintial step is set to 95°C for 2 minutes to allow polymerase to activate. Then, at 95°C for 30 seconds, denaturing takes place. The double strand DNA separates into single strands. Primers then anneal to complementary matches on the target DNA sequence when the temperature is at 57°C for 30 seconds. In the step for extending in the thermal cycle, the Taq polymerase adds nucleotides to extend the new DNA strand and it falls off after completion. When the tempeature is at 72°C for 2 minutes the polymerase finishes reading whatever strand they are currently on. For the final hold the temperature is at 4°C to help conserve the DNA by making sure everything "holds" so that it can later be analyzed or used.
Base-Pairing
"Base-pairing, driven by hydrogen bonding, allows base pairs to stick together". This allows dNTP's to combine, adenine(A) anneals to thymine(T), and cytosine(C) anneals to guanine(G).
Base-Pairing in Thermal Cycling
Base-pairing occurs during thermal cycling twice. It occurs during the anneal and extension step. In the anneal step, the primers have to bind to the single-strand DNA by combining with the complimentary nucleotides. In the extension step the nucleotides that are added by the Taq polymerase are involved in base-pairing also because in order for the single DNA strand to bind with the new one it would have to provide the complimentary nucleotides.
Bonus
SNP Information & Primer Design
Background: About the Disease SNP
The single nucleotide polymorphism, rs721710 is a polymorphism with a T/A origin that occurs in the 12th Chromosome of homo sapiens. A polymorphism is the affect of nucleotides, or the genetic building blocks of DNA, being altered compared to their natural form. The clinical significance of this single nucleotide polymorphism is unknown. According to a Pubmed, this polymorphism has been tested to determine the susceptibility a person has to Parkinson's disease. Many people with alterations to this gene are diagnosed with the disease.
Primer Design and Testing
The name of the gene LRRK2 stands for Leucine-rich Repeat Kinase. This gene contains instructions to for the enzyme dardarin. This is considered leucine rich as certain segments of this gene have a decent amount of this protein. This protein is important as it helps with interactions between other proteins or systems. This could be anything from sending signals all the way to helping cells make the cytoskeleton of the cell. The normal allele contains codon sequence GTG. An allele is the multiple forms a gene can take on due to mutations. The mutated allele codon sequence has an adenine nucleotide replacing the thymine nucleotide in the sequence. The position of this gene is 40,315,266th in the sequence.
Non Disease Forward Primer: 5'-TTAAGTGACTTGTACTTTGT-'3
Non Disease Reverse Primer: 5'-TGAAGCTCTTCAAGTAGTCT-'3
BME 100 Fall 2018
