LAB 4 WRITE-UP
- Lab coat and disposable gloves
- 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
- OpenPCR machine: shared by two groups
PCR Reaction Sample List
||PCR Reaction Sample
||Patient 1, replicate 1
||Patient 1, replicate 2
||Patient 1, replicate 3
||Patient 2, replicate 1
||Patient 2, replicate 2
||Patient 2, replicate 3
DNA Sample Set-up Procedure
- Start by moving the extracted DNA into a special PCR tube
- Label patients extracted DNA
- Next add Primer 1 to the PCR tube
- Now add Primer 2
- Add nucleotides to your PCR tube
- Add the DNA Polymerase to the PCR tube
- Place the PCR tube into a DNA thermal cycler
- 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
- 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 is the DNA strand of interest. We want to amplify part of the DNA sequence so it is easier to view.
- Primers: Attach to the part of the DNA sequence that you want to copy and amplify
- Taq Polymerase: Reads and replicates the DNA sequence of interest to create more copies. Taq polymerase is used in PCR because of its unique ability to withstand high temperatures.
- Deoxyribonucleotides (dNTP's): These are the building blocks of DNA that will be used to make copies of the template DNA
Q2. What happens to the components (listed above) during each step of thermal cycling?
- INITIAL STEP: 95°C for 2 minutes --> Preheats the DNA thermal cycler and establishes the desired temperature
- Denature at 95°C for 30 seconds: The double stranded DNA sequence falls apart resulting in two single stranded DNA sequences
- Anneal at 57°C for 30 seconds: Primers bind to their respective target sequences
- Extend at 72°C for 30 seconds: At this temperature Taq polymerase is activated. Polymerase finds the primers, attaches to the DNA single strands, and begins to add complimentary nucleotides to each sequence to create the new DNA strands.
- FINAL STEP: 72°C for 2 minutes --> Allows Taq polymerase to continue copying
- FINAL HOLD: 4°C --> The DNA is cooled to a very low temperature in order to stabilize the many new sequences
Q3. Base-pairing, driven by hydrogen bonding, allows base pairs to stick together. Which base anneals to each base listed below?
- Adenine (A): Binds to Thymine (T)
- Thymine (T): Binds to Adenine (A)
- Cytosine (C): Binds to Guanine (G)
- Guanine (G): Binds to Cytosine (C)
Q4. During which two steps of thermal cycling does base-pairing occur? Explain your answers.
- Annealing: The primers attach to the sequence of DNA that consists of their complimentary base pairs
- Extending: Taq Polymerase creates the new DNA strand by adding complimentary nucleotides to the existing strand
SNP Information & Primer Design
Background: About the Disease SNP
What is a nucleotide?
- The basic structural unit of DNA. It is a compound consisting of a nucleoside linked to a phosphate group.
What is a polymorphism?
- The presence of genetic variation within a population.
What species is this variation found in?
What chromosome is the variation located on?
What is listed as the Clinical Significance of this SNP?
What condition is linked to this SNP?
What does APOE stand for?
What is the function of APOE?
- Amyloid-beta binding
- Antioxidant activity
- Cholesterol binding
What is an allele?
- An alternate form of a gene that arises from mutation
The disease-associated allele contains what codon?
Primer Design and Testing
The numerical position of the SNP is:
Non-disease forward primer (20 nt):
The numerical position exactly 200 bases to the right of the disease SNP is:
Non-disease reverse primer (20 nt):
Disease forward primer (20 nt):
Disease reverse primer (20 nt):