Real-Time
PCR
BMO, P 3.1
Cheyenne van Kraaij
,Inhoud
Les 1a - Real time PCR ............................................................................................................................. 2
1.1 Difference between real-time PCR and end point PCR ................................................................. 2
1.2 Real-Time PCR principle ................................................................................................................ 2
1.3 Controls and aspecific amplification: PC/NTC/No-RT/IC, melting curves ..................................... 5
1.4 Quantification methods ................................................................................................................ 9
1.5 Detection: probes and fluorescent dyes ..................................................................................... 11
1.6 Mutation detection en overig ..................................................................................................... 20
Les 1b – Real-Time PCR ......................................................................................................................... 21
Les 2 – Real-Time-PCR ........................................................................................................................... 24
2.1 Analysis of PCR products ............................................................................................................. 24
2.2 Real-time PCR guidelines and terminology ................................................................................. 27
2.3 Real-time PCR quantification....................................................................................................... 30
Les 3 – Real-Time PCR ........................................................................................................................... 34
3.1 How to get control over the specificity of your PCR? ................................................................. 34
3.2 Getting control over your PCRs: What is PCR contamination? ................................................... 37
3.3 Two new controls in molecular diagnostics: no-RT and inhibition control ................................. 40
3.4 Aanvulling .................................................................................................................................... 46
Les 4 – Real-Time PCR ........................................................................................................................... 48
4.1 Interpretation of data from real-time PCR .................................................................................. 48
4.2 Some final pieces of theory ......................................................................................................... 50
Voorbereidende vragen ........................................................................................................................ 53
1
,Les 1a - Real time PCR
❖ Difference between real-time PCR and end point PCR
❖ Principle of real-time PCR
❖ Controls and aspecific amplification: PC/NTC/No-RT/IC, melting curves
❖ Quantification methods
❖ Detection: probes and fluorescent dyes
❖ Mutation detection
1.1 Difference between real-time PCR and end point PCR
Moleculaire diagnostiek: hoofdstukken 5 en 7
End point PCR or conventional PCR;
• PCR is stopped at a convenient cycle number to perform next steps e.g., gel electrophoresis or
sequencing
But what is the difference with real-time PCR?
• You can follow the PCR throughout all cycles using fluorescence measurements and you don’t
have to do the next steps required for end point PCR.
• Nowadays many diagnostic methods are based on real-time PCR
During real time PCR you can follow the DNA synthesis process on a PC
• When DNA synthesis is above background
• When exponential multiplication occurs
• When DNA synthesis ends on a plateau
1.2 Real-Time PCR principle
continuous monitoring of PCR product accumulation i.e. measure every cycle the amount of
fluorescence
relationship between the time fluorescence increases above background and the initial amount of
template i.e. the sooner fluorescence is visible, the more template was present, and vice versa
Bij realtime PCR wordt de toename van amplimeren die in opeenvolgende cycli worden
gesynthetiseerd afgelezen aan de stijging van een fluorescentie signal. Tijdens iedere cyclus wordt de
toenemende hoeveelheid fluorescentie gemeten. De meting vindt plaats aan het einde van de
annealingsfase (dual probes en molecular beacons) of wanneer de elongatiefase gereed is
(intercalerende kleurstoffen zoals SYBR Green, hydrolyse probes) (paragraaf 5.5)
R, Rn, ΔRn
R: fluorescence obtained without any normalization (raw data)
Rn: Normalized reporter signal = emission intensity of the reporter dye
emission intensity of the passive reference dye (ROX)
ΔRn = Rn – background (No Template Control; NTC)
2
, Rox: reduction of technical variation
ROX (carboxy-X-rhodamine) is used as a passive reference dye in ROX-dependent real-time PCR
systems to normalize for differences in fluorescence levels that can occur due mainly to optical path
variations among wells. ROX is not involved in the PCR reaction, and ROX fluorescence levels have no
relationship to the quantity of DNA in each well, so the addition of this fluorophore to a supermix
provides a constant fluorescent signal during the amplification cycles. The constancy of ROX
fluorescence relative to reporter fluorescence signal is used for well-to-well fluorescence
normalization.
Different types of real-time PCR systems that require a passive reference standard have different
optimal concentrations of ROX, mainly due to the different optical configurations of each system,
particularly the type of excitation source and optics used. For some PCR master mixes, the user must
determine the correct ROX concentration to optimize real-time PCR results; however, using Bio-Rad
universal real-time PCR supermixes, there is no guesswork — our universal reagents can be used on
any real-time PCR system.
To normalize real-time PCR data, the fluorescence emission intensity of the reporter dye (for
example, SYBR®Green) is divided by the fluorescence emission intensity of the ROX passive reference
dye. This ratio is the normalized reporter intensity, or Rn.
Amplification Plot and quantification Cycle, Cq.
Bij realtime PCR wordt de toename van amplimeren die in opeenvolgende cycli worden
gesynthetiseerd afgelezen aan de stijging van een fluorescentie signal. Tijdens iedere cyclus wordt de
toenemende hoeveelheid fluorescentie gemeten. De meting vindt plaats aan het einde van de
annealingsfase (dual probes en molecular beacons) of wanneer de elongatiefase gereed is
(intercalerende kleurstoffen zoals SYBR Green, hydrolyse probes)
3
PCR
BMO, P 3.1
Cheyenne van Kraaij
,Inhoud
Les 1a - Real time PCR ............................................................................................................................. 2
1.1 Difference between real-time PCR and end point PCR ................................................................. 2
1.2 Real-Time PCR principle ................................................................................................................ 2
1.3 Controls and aspecific amplification: PC/NTC/No-RT/IC, melting curves ..................................... 5
1.4 Quantification methods ................................................................................................................ 9
1.5 Detection: probes and fluorescent dyes ..................................................................................... 11
1.6 Mutation detection en overig ..................................................................................................... 20
Les 1b – Real-Time PCR ......................................................................................................................... 21
Les 2 – Real-Time-PCR ........................................................................................................................... 24
2.1 Analysis of PCR products ............................................................................................................. 24
2.2 Real-time PCR guidelines and terminology ................................................................................. 27
2.3 Real-time PCR quantification....................................................................................................... 30
Les 3 – Real-Time PCR ........................................................................................................................... 34
3.1 How to get control over the specificity of your PCR? ................................................................. 34
3.2 Getting control over your PCRs: What is PCR contamination? ................................................... 37
3.3 Two new controls in molecular diagnostics: no-RT and inhibition control ................................. 40
3.4 Aanvulling .................................................................................................................................... 46
Les 4 – Real-Time PCR ........................................................................................................................... 48
4.1 Interpretation of data from real-time PCR .................................................................................. 48
4.2 Some final pieces of theory ......................................................................................................... 50
Voorbereidende vragen ........................................................................................................................ 53
1
,Les 1a - Real time PCR
❖ Difference between real-time PCR and end point PCR
❖ Principle of real-time PCR
❖ Controls and aspecific amplification: PC/NTC/No-RT/IC, melting curves
❖ Quantification methods
❖ Detection: probes and fluorescent dyes
❖ Mutation detection
1.1 Difference between real-time PCR and end point PCR
Moleculaire diagnostiek: hoofdstukken 5 en 7
End point PCR or conventional PCR;
• PCR is stopped at a convenient cycle number to perform next steps e.g., gel electrophoresis or
sequencing
But what is the difference with real-time PCR?
• You can follow the PCR throughout all cycles using fluorescence measurements and you don’t
have to do the next steps required for end point PCR.
• Nowadays many diagnostic methods are based on real-time PCR
During real time PCR you can follow the DNA synthesis process on a PC
• When DNA synthesis is above background
• When exponential multiplication occurs
• When DNA synthesis ends on a plateau
1.2 Real-Time PCR principle
continuous monitoring of PCR product accumulation i.e. measure every cycle the amount of
fluorescence
relationship between the time fluorescence increases above background and the initial amount of
template i.e. the sooner fluorescence is visible, the more template was present, and vice versa
Bij realtime PCR wordt de toename van amplimeren die in opeenvolgende cycli worden
gesynthetiseerd afgelezen aan de stijging van een fluorescentie signal. Tijdens iedere cyclus wordt de
toenemende hoeveelheid fluorescentie gemeten. De meting vindt plaats aan het einde van de
annealingsfase (dual probes en molecular beacons) of wanneer de elongatiefase gereed is
(intercalerende kleurstoffen zoals SYBR Green, hydrolyse probes) (paragraaf 5.5)
R, Rn, ΔRn
R: fluorescence obtained without any normalization (raw data)
Rn: Normalized reporter signal = emission intensity of the reporter dye
emission intensity of the passive reference dye (ROX)
ΔRn = Rn – background (No Template Control; NTC)
2
, Rox: reduction of technical variation
ROX (carboxy-X-rhodamine) is used as a passive reference dye in ROX-dependent real-time PCR
systems to normalize for differences in fluorescence levels that can occur due mainly to optical path
variations among wells. ROX is not involved in the PCR reaction, and ROX fluorescence levels have no
relationship to the quantity of DNA in each well, so the addition of this fluorophore to a supermix
provides a constant fluorescent signal during the amplification cycles. The constancy of ROX
fluorescence relative to reporter fluorescence signal is used for well-to-well fluorescence
normalization.
Different types of real-time PCR systems that require a passive reference standard have different
optimal concentrations of ROX, mainly due to the different optical configurations of each system,
particularly the type of excitation source and optics used. For some PCR master mixes, the user must
determine the correct ROX concentration to optimize real-time PCR results; however, using Bio-Rad
universal real-time PCR supermixes, there is no guesswork — our universal reagents can be used on
any real-time PCR system.
To normalize real-time PCR data, the fluorescence emission intensity of the reporter dye (for
example, SYBR®Green) is divided by the fluorescence emission intensity of the ROX passive reference
dye. This ratio is the normalized reporter intensity, or Rn.
Amplification Plot and quantification Cycle, Cq.
Bij realtime PCR wordt de toename van amplimeren die in opeenvolgende cycli worden
gesynthetiseerd afgelezen aan de stijging van een fluorescentie signal. Tijdens iedere cyclus wordt de
toenemende hoeveelheid fluorescentie gemeten. De meting vindt plaats aan het einde van de
annealingsfase (dual probes en molecular beacons) of wanneer de elongatiefase gereed is
(intercalerende kleurstoffen zoals SYBR Green, hydrolyse probes)
3
