20.02.09 Quantitative real time PCR

Question 1

What is quantitative PCR

Answer 1

• 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.

Question 2

Which phase of the reaction is the amount of product produced directly proportional to amount of template

Answer 2

Exponential phase

Question 3

How many PCR products are produced with every cycle if efficiency is 100%

Answer 3

Products double with every cycle

Question 4

Which part of the reaction is used for accurate quantification

Answer 4

Exponential

Question 5

What is reverse transcription qPCR

Answer 5

When cDNA is used as a template

Question 6

What is used to internally calibrate gene expression of a transcript of interest

Answer 6

A reference gene

Question 7

What are the MIQE guidelines

Answer 7

• Minimum information for publication of Quantitative digital PCR Experiments.
• Published 2009

Question 8

What two methods are used to detect PCR products in qPCR

Answer 8

1. non-specific fluorescent dyes that intercalate with any dsDNA (SYBR green, fluoresces strongly when it binds to minor groove of dsDNA)
2. Sequence-specific DNA probes. Oligonucleotides labelled with a fluorescent reporter. e.g. Taqman hydrolysis probes, FRET hybridisation probes or Molecular Beacon hairpin probes.

Question 9

Explain Taqman assay

Answer 9

• 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)

Question 10

Explain FRET hybridisation assay

Answer 10

• 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)

Question 11

Explain Molecular Beacon hairpin probes

Answer 11

• 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.

Question 12

Pros of non-specific intercalating agents

Answer 12

• Can monitor the amplification of any dsDNA sequence
• No probe required, reduces assay set up and cost
• Simple, can be used for any reaction

Question 13

Cons of non-specific intercalating agents

Answer 13

• 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

Question 14

Pros of sequence specific assays

Answer 14

• 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

Question 15

Cons of sequence specific assays

Answer 15

• Multiple reactions required for multiple loci
• Different probes must be synthesised for each unique target sequence
• probes are expensive.

Question 16

Which phase of reaction can quantification be done accurately

Answer 16

exponential or log linear phase

Question 17

What is the cycle threshold (Ct)

Answer 17

-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.

Question 18

Ct values are inversely proportional to what

Answer 18

• the amount of template in the sample

- The lower the Ct value the greater the amount of template.

Question 19

What factors impact Ct values

Answer 19

• 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.

Question 20

What is the baseline

Answer 20

The limit of detection of the instrument, cannot be distinguished from background noise.

Question 21

What is the threshold

Answer 21

Level of signal that reflects a statistically significant increase over the calculated baseline signal. Distinguishes relevant amplification from background.

Question 22

What is DeltaRN

Answer 22

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.

Question 23

What two types of quantification are there

Answer 23

• Absolute quantification

- Relative quantification

Question 24

What is absolute quantification

Answer 24

• 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.

Question 25

What is relative quantification

Answer 25

• 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.

Question 26

Give 5 examples of applications of qPCR in diagnostics.

Answer 26

1. Counting bacterial, viral or fungal load.
2. Monitoring of neoplastic disorders.
3. Single base mutation detection
4. SNP genotyping
5. Genomic Copy number measurement

Question 27

Explain the use of qPCR in counting bacterial, viral or fungal load.

Answer 27

• 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

Question 28

Explain the use of qPCR in monitoring of neoplastic disorders

Answer 28

• 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.

Question 29

Explain the use of qPCR in single base mutation detection

Answer 29

• 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.

Question 30

Explain the use of qPCR in SNP genotyping

Answer 30

• Two labelled probes with different fluorophores. One specific for each allele.
• Compare fluorescence

Question 31

Explain the use of qPCR in Genomic Copy Number Measurement

Answer 31

• Used to confirm array findings as an alternative to FISH.
• Doesn’t provide positional information.
• Useful to confirm small imbalances.