PCR reac on 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
Cup for discarded ps
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
49652
G3 1-2
Patient 1, replicate 2
49652
G3 1-3
Patient 1, replicate 3
49652
G3 2-1
Patient 2, replicate 1
99085
G3 2-2
Patient 2, replicate 2
99085
G3 2-3
Patient 2, replicate 3
99085
DNA Sample Set-up Procedure
Step 1: Get two patient ID numbers from a Teaching Assistant and have them written down.
Step 2: In the report labels will be placed on each tube to identify what specimen is in which tube. For Group 3, these tubes will be marked as G3 P, G3 N, G3 1-1, and etc.
Step 3: The results will then be displayed in a graph which contains all specimen.
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:
Provides optimal input amounts for PCR amplification
Primers:
Bind to 3' ends of the DNA where the desired template is
Taq Polymerase:
Replicates the target DNA
Deoxyribonucleotides:
Work as building blocks for the new DNA strands
Q2. What happens to the components (listed above) during each step of thermal cycling?
INITIAL STEP: 95°C for 2 minutes:
The DNA separated due to the heat
Denature at 95°C for 30 seconds:
Strands are unpaired
Anneal at 57°C for 30 seconds:
Primers bind to the target DNA sequences
Extend at 72°C for seconds:
Taq DNA polymerase binds
FINAL STEP: 72°C for 2 minutes:
Begins adding nucleotides to the DNA
FINAL HOLD: 4°C
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?
Andenine (A) pairs with:
Thymine(T)
Thymine(T) pairs with:
Andenine (A)
Cytosine (C) pairs with:
Guanine (G)
Guanine (G) pairs with:
Cytosine (C)
Q4. During which two steps of thermal cycling does base‐pairing occur? Explain your answers.
During the second step of thermal cycling the TAQ DNA polymerase begin to add nucleotides to the template DNA strands
Part 1: Use the NCBI database to find a disease-associated sequence
What is a nucleotide?
small-scale multi-base deletions or insertions (also called deletion insertion polymorphisms or DIPs)
What is a polymorphism?
A common variation in the sequence of DNA among individuals. Genetic variations occurring in more than 1% of the population would be considered useful polymorphisms for genetic linkage analysis.
What species is this variation found in?
Homo sapiens
What chromosome is the variation located on?
12:40315266
What is listed as the Clinical significance of this SNP?
Uncertain significance
What condition is linked to this SNP?
Susceptibility to Parkinson’s disease
Part 2 : Find the DNA sequence of the SNP, and the surrounding sequence
What does LRRK2 stand for?
Leucine rich repeat kinase 2
What is the function of LRRK2?
Protein binding, Clathrin binding, MAP kinase kinase activity
What is an allele?
One of two or more alternative forms of a gene that arise by mutation and are found at the same place on a chromosome.
The disease-associated allele contains what codon?
GAG
What numerical position of the SNP?
Position: 40315266
Part 3: Now that you have a good view of the DNA sequence, you can design primer for PCR
Non-disease forward primer (20 nt)?
5'-TTAAGTGACTTGTACTTTGT-3'
the numerical position exactly 20 bases to the right of the disease SNP is?
40315466
Non-disease forward primer (20 nt)?
5'-TGAAGCTCTTCAAGTAGTCT-3'
Disease forward primer (20 nt)?
5'-TTAAGTGACTTGTACTTTGA-3'
Disease reverse primer (20 nt)?
5'-TGAAGCTCTTCAAGTAGTCT-3'
SNP Information & Primer Design
Background: About the Disease SNP
SNP stands for Single Nucleotide Polymorphisms, in which occurs in every 300 nucleotides on average. There are nearly 10 million SNPS in the human genome that plays a more direct role in disease by affecting the genes function. It has recently been proven that SNPS has help identify the response of the human body with certain drug uses. As well as tracing inheritance of disease within families. Overall, SNPS is a programmed bio-marker to the human use.
Primer Design and Testing
In the primer test the results are the following.In the nucleotide sequence, variations are used as positional markers. When placed in a unique location in a genome, the marker works with the same logic as a sequence STSs or framework microsatlellite markers. In other words the flake sequence services as a landmark in the genome. As shown in the figure below the flake base either assayed for variation.
BME 100 Fall 2018
