QPCR Ai Flashcards
What is qPCR?
qPCR, also known as real-time PCR, is a laboratory technique used to amplify and quantify DNA.
How does qPCR differ from regular PCR?
Unlike regular PCR, which only amplifies DNA, qPCR allows you to monitor the amount of DNA being produced in real time during each cycle.
What components are used in qPCR for DNA amplification?
- Primers
- DNA polymerase
- Nucleotides
What is the role of fluorescent detection in qPCR?
qPCR uses fluorescent dyes or probes that bind to the DNA, increasing fluorescence as DNA is amplified.
What machine is used to detect fluorescence in qPCR?
qPCR thermocycler
What are the two main types of qPCR assays?
- SYBR Green qPCR
- TaqMan Probe qPCR
What does the amplification curve in qPCR show?
The amplification curve shows how the fluorescence signal increases as the DNA is amplified over multiple cycles.
What are the four distinct phases of the amplification curve in qPCR?
- Baseline (LAG) Phase
- Exponential Phase
- Linear (Log-Linear) Phase
- Plateau Phase
What happens during the Baseline (LAG) Phase of qPCR?
Early cycles of PCR; the amount of DNA is still very low and the fluorescence signal is too low to be detected.
What occurs in the Exponential Phase of qPCR?
DNA is doubling each cycle with high efficiency, and the fluorescence signal rises rapidly above the baseline.
What is the Cq value in qPCR?
The cycle number during a qPCR run where the fluorescence signal first rises above the background threshold.
How does the Cq value relate to the amount of starting DNA?
The lower the Cq value, the more starting DNA was present in the sample.
List some applications of qPCR.
- Detecting and quantifying pathogens
- Gene expression analysis
- DNA quantification
- Mutation detection
- Diagnostic tests
What is the first step in a qPCR protocol?
Sample Preparation: Extract DNA or RNA from your biological sample.
What is SYBR Green qPCR?
Uses a dye that binds to any double-stranded DNA.
What is TaqMan Probe qPCR?
Uses a sequence-specific fluorescent probe for more specificity.
Describe the process of the SYBR Green Assay.
- Denaturation (95°C): Double-stranded DNA melts into single strands.
- Annealing: Primers attach to their target sequences.
- Extension: DNA polymerase builds new strands, forming dsDNA again.
What is Melt Curve Analysis used for in qPCR?
Determines melting temperature to verify product specificity.
How does the TaqMan Assay work?
Uses a short DNA probe with a reporter dye and a quencher; fluorescence is released when the probe is cleaved during PCR.
What are the main differences between TaqMan and Lux detection?
- TaqMan: High specificity, more complex, more expensive
- Lux: Moderate specificity, simpler, less expensive
What instruments are used in Real-Time PCR?
- Thermal Cycler
- Optical Module
- Computer with Software
What does the Optical Module do in a qPCR machine?
Detects fluorescence in each tube or well during the PCR run.
What is the purpose of including no-template controls (NTC) in a qPCR experiment?
To check for contamination.
True or False: SYBR Green can detect non-specific products like primer-dimers.
True
Fill in the blank: The amplification curve in qPCR consists of four phases: Baseline, __________, Linear (Log-Linear), and Plateau.
Exponential Phase
