20.02.09 Quantitative real time PCR Flashcards
What is quantitative PCR
- Simultaneous amplification, detection and quantification of DNA templates during each PCR cycle in real time.
- Fluorescence is directly proportional to the amount of amplified product.
Which phase of the reaction is the amount of product produced directly proportional to amount of template
Exponential phase
How many PCR products are produced with every cycle if efficiency is 100%
Products double with every cycle
Which part of the reaction is used for accurate quantification
Exponential
What is reverse transcription qPCR
When cDNA is used as a template
What is used to internally calibrate gene expression of a transcript of interest
A reference gene
What are the MIQE guidelines
- Minimum information for publication of Quantitative digital PCR Experiments.
- Published 2009
What two methods are used to detect PCR products in qPCR
- non-specific fluorescent dyes that intercalate with any dsDNA (SYBR green, fluoresces strongly when it binds to minor groove of dsDNA)
- Sequence-specific DNA probes. Oligonucleotides labelled with a fluorescent reporter. e.g. Taqman hydrolysis probes, FRET hybridisation probes or Molecular Beacon hairpin probes.
Explain Taqman assay
- Uses 5’ to 3’ exonuclease activity of TaqDNA polymerase
- Taq probes are fluorescently labelled at their 5’ end (e.g. with FAM) and have a quencher (e.g. TAMRA) at 3’ end. When intact the quencher is in close proximity to the fluorescent tag so signal is quenched. DUe to FRET (fluorescence resonance energy transfer).
- During PCR Taq pol cleaves an oligonucleotide probe releasing the quencher, allowing dye to fluoresce. Signal is then detected.
- Probes are specific for minor groove (gives specificity and allows for a smaller probe)
Explain FRET hybridisation assay
- Uses a pair of fluorescent probes (donor probe labelled at 3’ and acceptor probe at 5’)
- When primers are bound the energy is transferred between the two fluorophores leading to a FRET-based increase in fluorescence emitted.
- Once PCR extension occurs the donor probe is cleaved, so energy transfer stops and emission stops.
- Can be used for melt curve analysis (hydrolysis probes can’t)
Explain Molecular Beacon hairpin probes
- Similar to hydrolysis probes, they have fluorophore at 5’ and quencher at 3’ end.
- Complementary ends so they form a hairpin structure.
- In the presence of complementary target sequence the probe unfolds and hybridises to target sequence. This causes the fluorophore to be displaced from the quencher produces signal.
Pros of non-specific intercalating agents
- Can monitor the amplification of any dsDNA sequence
- No probe required, reduces assay set up and cost
- Simple, can be used for any reaction
Cons of non-specific intercalating agents
- Not specific, will bind to any dsDNA (target or non-target). Could lead to false positive signals.
- Primer dimers are an issue
- Multiplexing less achievable
- Melting curve analysis required for accuracy
Pros of sequence specific assays
- Specific hybridisation between probe and target required to generate a signal
- Probes can be labelled with different reporters dyes to allow amplification of two distinct sequences in one reaction
- Small amplicon size results in increased amplification efficiency (even with degraded DNA)
- Post-PCR processing is eliminated, reducing labour and material costs
Cons of sequence specific assays
- Multiple reactions required for multiple loci
- Different probes must be synthesised for each unique target sequence
- probes are expensive.
Which phase of reaction can quantification be done accurately
exponential or log linear phase
What is the cycle threshold (Ct)
-Number of cycles required for the fluorescent signal to be detected above the background/ baseline level, after which the exponential increase is seen and quantification can occur.
Ct values are inversely proportional to what
- the amount of template in the sample
- The lower the Ct value the greater the amount of template.
What factors impact Ct values
- Template concentration (higher the starting conc, the fewer the cycles required to reach Ct threshold)
- PCR efficiency
- Reaction mix.
- Therefore can’t directly compare runs that used different conditions or reagents.
What is the baseline
The limit of detection of the instrument, cannot be distinguished from background noise.
What is the threshold
Level of signal that reflects a statistically significant increase over the calculated baseline signal. Distinguishes relevant amplification from background.
What is DeltaRN
An increment of fluorescent signal at each time point. Reporter signal minus the baseline level. Values are plotted on y axis with cycle number on x axis. Should form a sigmoid curve.
What two types of quantification are there
- Absolute quantification
- Relative quantification
What is absolute quantification
- Relates PCR signal to input copy number using a calibration curve
- Calibration curve created using a set of dilution standards of known concentration.
- Test sample is plotted against the log of the standard concentration to determine it’s concentration
- Reliability depends on the condition of identical amplification efficiencies between standard and test sample.
What is relative quantification
- An internal reference gene is used to determine the fold-differences in expression levels of the target gene.
- Easier than absolute, as a calibration curve is not necessary, so eliminates dilution errors made when creating the curve.
- Efficiency of the target and reference PCR must be equal.
Give 5 examples of applications of qPCR in diagnostics.
- Counting bacterial, viral or fungal load.
- Monitoring of neoplastic disorders.
- Single base mutation detection
- SNP genotyping
- Genomic Copy number measurement
Explain the use of qPCR in counting bacterial, viral or fungal load.
- Measures the presence and quantity of pathogenic-specific sequences within a sample.
- Faster than culturing
- Used to measure disease progression and efficacy of antiviral treatment
Explain the use of qPCR in monitoring of neoplastic disorders
- qPCR used to measure disease-specific prognostic markers of minimal residual disease.
- More sensitive than other cytogenetic techniques
- CML. BRC-ABL1 transcript leels used for early detection of relapse of CML.
- ALL. Used to monitor gene fusions (BRC-ABL1, MLL-AF4) or if absent a rearrangement of immunoglobulin or T-cell receptor genes.
- AML. Used to monitor gene fusions (PML-RARA, RUNX1-RUNXT1), mutations and transcripts that are unregulated in AML (e.g. WT1).
- Used for quantitative assessment of haematopoietic chimerism after bone marrow transplantation.
Explain the use of qPCR in single base mutation detection
- Used to detect presence of a known mutation.
- Quenched FRET based assay, where decrease in fluorescence of the donor fluorophore is measured each cycle. Melt curve analysis is then performed to distinguish between wild-type and mutant alleles.
Explain the use of qPCR in SNP genotyping
- Two labelled probes with different fluorophores. One specific for each allele.
- Compare fluorescence
Explain the use of qPCR in Genomic Copy Number Measurement
- Used to confirm array findings as an alternative to FISH.
- Doesn’t provide positional information.
- Useful to confirm small imbalances.
