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: only use each only once. Never reuse disposable pipette tips. If you do, the samples will become cross‐contaminated
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
46547
G4 1-2
Patient 1, replicate 2
46547
G4 1-3
Patient 1, replicate 3
46547
G4 2-1
Patient 2, replicate 1
41967
G4 2-2
Patient 2, replicate 2
41967
G4 2-3
Patient 2, replicate 3
41967
DNA Sample Set-up Procedure
Step 1: DNA extracted from a subject is placed into a PCR that evenly distributes heat required for PCR.
Step 2: The first primer is then added to the PCR tube.
Step 3: The second primer is also added to the PCR tube, and during the PCR process the primers will attach to either end of the DNA strand.
Step 4: Next Nucleotides (A,T,C,G) are added to the PCR tube.
Step 5: DNA Polymerase is the last component added to the PCR tube.
Step 6: Lastly the PCR tube/ tubes are put into the thermal cycler to run through the PCR process.
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
Q1. What is the function of each component of a PCR reaction?
Template DNA:
This contains the gene that needs to be replicated in this process.
Primers:
They tag the DNA templates where the desired genes are so that the proper sections can be replicated.
Taq Polymerase:
This enzyme assembles the nucleotides on the template DNA so that it is replicated.
Deoxyribonucleotides (dNTPs):
These make replication of the DNA template possible by providing the base pairs that create the DNA molecules themselves.
Q2. What happens to the components during each step of thermal cycling?
INITIAL STEP: 95°C for 2 minutes:
DNA is attempting to reach an optimal temp of 95°C. DNA strand uncoils.
Denature at 95°C for 30 seconds:
DNA strands separate into two single strand DNA molecules.
Anneal at 57°C for 30 seconds:
Primers cling to DNA strands before they join reattach. Polymerase copies specified DNA sequence.
Extend at 72°C for 30 seconds:
Taq polymerase attaches itself to single DNA strand.
FINAL STEP: at 57°C for 2 minutes:
Polymerase reads DNA and attaches corresponding nucleotides.
FINAL HOLD: 4°C:
The replication process is completed and DNA copies are stabilized.
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
Guanin (G)
C
Q4. During which two steps of thermal cycling does base-pairing occur?
Base pairing occurs during the annealing (57 degrees Celsius) process when the primers and their bases pair with the desired sections of DNA. Base pairing also occurs during the extension (72 degrees Celsius) process, when the DNA polymerase binds to the primers and begins adding base pairs to the DNA strand.
PCR Lab B
Section 1: Disease SNP-Specific Primer Design
Goal: Learn how PCR can be applied to detect a disease-associated DNA sequence (SNP). You will learn by completing the process of designing an SNP-specific primer pair.
Part 1:
What is a nucleotide?
A nucleotide is the monomer of a nucleic acid polymer. It is the basic structural unit of DNA and RNA.
What is a polymorphism?
Polymorphisms are different forms of a single gene that code for different discrete expressions of the gene, such as blood type.
What species is this variation found? (latin name)
Homo sapiens
What chromosome is the variation located on?
12:40315266
What is listed as the Clinical Significance of this SNP?
Clinical significance is uncertain.
What condition is linked to the SNP?
It is linked to Parkinson’s disease.
Part 2
What does LRRK2 stand for?
It stands for leucine rich repeat kinase 2.
Whatis the function of LRRK2?
It is responsible for ATP binding, GTP binding, and GTP-dependent protein kinase activity.
What is an allele?
An allele is one of the two possible forms of a gene in an organism and are found on a certain position of a chromosome.
The disease-associated allele contains what codon?
It contains the codon GAG.
The numerical position of the SNP is:
The numerical position of the SNP is 40315266.
Part 3
Non-disease forward primer (20 nt):
5’-TTAAGTGACTTGTACTTTGT-3’
The numerical position exactly 200 bases to the right of the disease SNP is:
40315466
Non-disease reverse primer (20 nt):
5’-TGAAGCTCTTCAAGTAGTCT-3’
Disease forward primer (20 nt):
5’-TTAAGTGACTTGTACTTTGA-3’
Disease revers primer (20 nt):
5’-TGAAGCTCTTCAAGTAGTCT-3’
SNP Information & Primer Design
Background: About the Disease SNP
A Single nucleotide polymorphism (SNP) is a variation of a specific base pair (A/T or G/C) within DNA. Variations of SNP's effect a single nucleotide and can occur with every 200 to 300 nucleotides or base pairs within a DNA sequence. The significance of SNP's in relation to diseases can reveal why certain pharmaceuticals have negative effects on patients or the probability to develop hereditary diseases. The disease SNP rs721710 was used for Lab B and is linked to Parkinson's disease, although the clinical significance was uncertain. This specific SNP is located on chromosome 12:40315266. LRRK2 or leucine rich repeat kinase 2 is the gene that contains the SNP used in Lab B and is comprised of multiple base pairs that occupy a specific area of a chromosome.
Primer Design and Testing
The Non-Disease Forward Primer: 5’-TTAAGTGACTTGTACTTTGT-3’
The numerical position exactly 200 bases to the right of the disease SNP is 40315466.
The Non-Disease reverse primer: 5’-TGAAGCTCTTCAAGTAGTCT-3’
Disease Forward Primer: 5’-TTAAGTGACTTGTACTTTGA-3’
Disease Reverse Primer: 5’-TGAAGCTCTTCAAGTAGTCT-3’
BME 100 Fall 2018
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