FRSC 438 Test 2 Questions With Complete
Answers 100% Pass
What is PCR - ANSWER Enzymatic reaction to amplify a specific region of DNA
sequence
What are the cycles of PCR - ANSWER 1. Denaturation
2. Annealing
3. Extension
Denaturation - ANSWER Done at 95C to break the H bonds between bases and creating
ssDNA
Annealing - ANSWER Done at 55-65C where DNA premiers anneal and flank locus. of
interest
Extension - ANSWER Done at 70-72C so DNA polymers extends primer at 3' end to
create complimentary strand
How much template DNA do you need in a reaction? - ANSWER 0.5 to 1ng
What strand does the forward primer bind to? Reverse Primer? - ANSWER Forward:
Minus strand
Reverse: Plus strand
List primer design and selection - ANSWER - unique sequence 18-30bp
- Flank known sequence (100-600bp)
- Avoid Poly DNTP
- 50% G/C content
What can cause complimentary primer pair sequences - ANSWER - primer dimer
- hairpin loop
PCR specificity is determined by... - ANSWER Primers and Tm
Higher Tm increases specificity
What is the role of MgCl2 in PCR? - ANSWER Cofactor to Taq polymerase
What is the role of the buffer in PCR? - ANSWER Maintains optimal pH and salt
, concentration for enzymatic activity
How do you prevent premature polymerase activity? - ANSWER 1. Add taq last
2. Keep all PCR reactions on ice
3. Use a hot start polymerase (95C for 10min)
How do yo prevent non-template adenylation? - ANSWER Add 30-90 min final extension
step at end of PCR run to ensure consistent amount of A's added to each PCR product
What causes non-unique amplification products? What are potential causes? - ANSWER
Non-specific amplification
Causes: Too low annealing temp., poor primer selection, premature Taq activation
Advantages of PCR - ANSWER - fast and simple
- little DNA required
- Degraded DNA can be analyzed
- Loci analyzed simultaneously
- standardized results
Disadvantages to PCR? - ANSWER Each locus has low power of discrimination
What is qPCR? - ANSWER quantitative PCR or 'real-time' quantitative PCR:
• Used to quantify the abundance of a particular DNA sequence in a sample. (Human
specific)
• Uses fluorescence to measure amplification of target sequence as it happens, cycle by
cycle (the rate of PCR amplification is related to target abundance in the sample being
used as template)
What are the qPCR phases? - ANSWER Phase 1: linear-ground (baseline)
Phase 2: Exponential (increase signal proportionate to PCR product)
Phase 3: Linear (1+ PCR components are rate limiting, Threshold)
Phase 4: Plateau (PCR stops)
QPCR Taqman probe - ANSWER Reporter dye linked to 5' end and non-fluorescent
quencher at 3' end
Förster resonance energy transfer - ANSWER In close proximity, energy from donor is
transferred to acceptor molecule rather than being emitted as light
(Dipole-Dipole coupling)
Answers 100% Pass
What is PCR - ANSWER Enzymatic reaction to amplify a specific region of DNA
sequence
What are the cycles of PCR - ANSWER 1. Denaturation
2. Annealing
3. Extension
Denaturation - ANSWER Done at 95C to break the H bonds between bases and creating
ssDNA
Annealing - ANSWER Done at 55-65C where DNA premiers anneal and flank locus. of
interest
Extension - ANSWER Done at 70-72C so DNA polymers extends primer at 3' end to
create complimentary strand
How much template DNA do you need in a reaction? - ANSWER 0.5 to 1ng
What strand does the forward primer bind to? Reverse Primer? - ANSWER Forward:
Minus strand
Reverse: Plus strand
List primer design and selection - ANSWER - unique sequence 18-30bp
- Flank known sequence (100-600bp)
- Avoid Poly DNTP
- 50% G/C content
What can cause complimentary primer pair sequences - ANSWER - primer dimer
- hairpin loop
PCR specificity is determined by... - ANSWER Primers and Tm
Higher Tm increases specificity
What is the role of MgCl2 in PCR? - ANSWER Cofactor to Taq polymerase
What is the role of the buffer in PCR? - ANSWER Maintains optimal pH and salt
, concentration for enzymatic activity
How do you prevent premature polymerase activity? - ANSWER 1. Add taq last
2. Keep all PCR reactions on ice
3. Use a hot start polymerase (95C for 10min)
How do yo prevent non-template adenylation? - ANSWER Add 30-90 min final extension
step at end of PCR run to ensure consistent amount of A's added to each PCR product
What causes non-unique amplification products? What are potential causes? - ANSWER
Non-specific amplification
Causes: Too low annealing temp., poor primer selection, premature Taq activation
Advantages of PCR - ANSWER - fast and simple
- little DNA required
- Degraded DNA can be analyzed
- Loci analyzed simultaneously
- standardized results
Disadvantages to PCR? - ANSWER Each locus has low power of discrimination
What is qPCR? - ANSWER quantitative PCR or 'real-time' quantitative PCR:
• Used to quantify the abundance of a particular DNA sequence in a sample. (Human
specific)
• Uses fluorescence to measure amplification of target sequence as it happens, cycle by
cycle (the rate of PCR amplification is related to target abundance in the sample being
used as template)
What are the qPCR phases? - ANSWER Phase 1: linear-ground (baseline)
Phase 2: Exponential (increase signal proportionate to PCR product)
Phase 3: Linear (1+ PCR components are rate limiting, Threshold)
Phase 4: Plateau (PCR stops)
QPCR Taqman probe - ANSWER Reporter dye linked to 5' end and non-fluorescent
quencher at 3' end
Förster resonance energy transfer - ANSWER In close proximity, energy from donor is
transferred to acceptor molecule rather than being emitted as light
(Dipole-Dipole coupling)
