Real-time PCR Optics
An integrated fluorimeter is required to detect and monitor the levels of fluorescence during the PCR process for real-time PCR and there is a range of options available for both the excitation light source and fluorescent emission detection. The light sources that cause fluorophore excitation can be classed as narrow- or broad-spectrum. If a broad-spectrum light source is employed (e.g. Mx4000, Mx3000P, Mx3005P, MyiQ, iQ5, ABI 7300, ABI 7500, LightCycler 480) then filters can be used to provide light tuned to the excitation spectrum of a specific individual fluorophore. Such a system provides the user with a wider choice of available fluorophores, although it is best to select those with good separation of their emission spectra.
A disadvantage of this optical system is that the light intensity passing through the filters can be limited and this could in theory limit the sensitivity of detection. There are currently two narrow-spectrum light sources used in real-time platforms, these can be light emitting diodes (LEDs) (e.g. LightCyclers 1.5 and 2.0, Opticon2, MiniOpticon, Chromo4, SmartCycler, Rotor-Gene, StepOne, Mastercycler ep) or laser (ABI 7900HT). The SmartCycler and Rotor-Gene have multiple LEDs (4 and 6 respectively) that excite at different wavelengths, providing a greater selection of fluorophores and giving these instruments capabilities similar to those of the broad-spectrum platforms above. The LightCyclers 1.5 & 2.0, Opticon2 and ABI 7900HT have single light source excitation, which ultimately limits the choice of fluorophores.
In general, the detectors used in real-time platforms are set to measure narrow bands of the spectrum, although filter sets that can be customised by the user are available for the iQ5, Mx4000, Mx3005P and Mx3000P, Chromo4. The number of detection channels that can be effective is dependent on the available range of excitation wavelengths. For example, if a single narrow range excitation source is available, one approach is to use fluorophores that are all excited to some extent in the same range and then to rely on software correction to deconvolute the light emitted from a given area of the spectrum, as was employed successfully with the now discontinued ABI 7700.
Another approach is demonstrated with the LightCycler 1.5, where a narrow-spectrum light source excites the fluorophores SYBR or fluorescein and emitted light is collected via three discrete optical detectors. Two of these detect long wavelength light emissions from fluorophores which are only minimally excited by the blue LED light source, which are instead excited using FRET technology.
from Logan and Edwards (2009) in Real-Time PCR: Current Technology and Applications
Bibliography:
A disadvantage of this optical system is that the light intensity passing through the filters can be limited and this could in theory limit the sensitivity of detection. There are currently two narrow-spectrum light sources used in real-time platforms, these can be light emitting diodes (LEDs) (e.g. LightCyclers 1.5 and 2.0, Opticon2, MiniOpticon, Chromo4, SmartCycler, Rotor-Gene, StepOne, Mastercycler ep) or laser (ABI 7900HT). The SmartCycler and Rotor-Gene have multiple LEDs (4 and 6 respectively) that excite at different wavelengths, providing a greater selection of fluorophores and giving these instruments capabilities similar to those of the broad-spectrum platforms above. The LightCyclers 1.5 & 2.0, Opticon2 and ABI 7900HT have single light source excitation, which ultimately limits the choice of fluorophores.
In general, the detectors used in real-time platforms are set to measure narrow bands of the spectrum, although filter sets that can be customised by the user are available for the iQ5, Mx4000, Mx3005P and Mx3000P, Chromo4. The number of detection channels that can be effective is dependent on the available range of excitation wavelengths. For example, if a single narrow range excitation source is available, one approach is to use fluorophores that are all excited to some extent in the same range and then to rely on software correction to deconvolute the light emitted from a given area of the spectrum, as was employed successfully with the now discontinued ABI 7700.
Another approach is demonstrated with the LightCycler 1.5, where a narrow-spectrum light source excites the fluorophores SYBR or fluorescein and emitted light is collected via three discrete optical detectors. Two of these detect long wavelength light emissions from fluorophores which are only minimally excited by the blue LED light source, which are instead excited using FRET technology.
from Logan and Edwards (2009) in Real-Time PCR: Current Technology and Applications
Bibliography:
- Real-Time PCR: Current Technology and Applications
- Real-Time PCR in Microbiology: From Diagnosis to Characterization
- PCR Troubleshooting: The Essential Guide
- PCR Books
Labels: ABI 7300, ABI 7500, ABI 7900HT, Chromo4, fluorimeter, iQ5, LightCycler, LightCycler 480, Mastercycler, MiniOpticon, Mx3000P, Mx3005P, Mx4000, MyiQ, Opticon2, PCR optics, Rotor-Gene, SmartCycler, StepOne
Real-time PCR Thermocycling
The first component to consider in a PCR platform is the thermal engine. Successful thermal cycling is dependent on the accurate regulation of temperature in the sample vessels and the speed at which these target temperatures can be achieved. The majority of real-time platforms use advanced heating block technology based on the Peltier-effect, to actively transfer heat in and out of thin-walled plastic reaction vessels (e.g. ABI 7300, ABI 7500, ABI 7900HT, ABI Step One, Opticon 2, MiniOpticon, Chromo 4, Mx4000, Mx3000P, Mx3005P, Mastercycler ep, MyiQ, iQ5, LightCycler 480).
Peltier devices transfer heat from one side of a semiconductor to another. In general, blocks have significant mass and consequently a degree of thermal inertia. Furthermore, the plastic insulating layer between the reaction vessel and the heater produces an additional thermal lag. As a consequence of this, the temperature transitions are relatively slow and blocks must be very carefully designed to minimise well-to-well variation. Other advances on the Peltier-based technology include its combination with Joule, resistive or convective technology to give improved temperature control and performance across the block.
More recently has been the inclusion of patented Therma-Base technology, whose working principle is based on the evaporation and condensation of a working fluid in a thin vacuum cavity to accurately control well-to-well variation. Three platforms employ alternative heat exchange technologies which permit more rapid thermal ramp rates than blocks, resulting in significantly increased thermocycling speeds. These include a stationary turbulent air-heated glass capillary format (LightCyclers 1.5 and 2.0), a centrifugal air-heated plastic tube format (Rotor-Gene) and a high-thermal-conductivity ceramic heating plate plastic tube format (SmartCycler). For example, the time taken to equilibrate at 72°C using a Rotor-Gene is 0s compared to 15s with a standard 96-well peltier block, resulting in run times that are on average 50% faster. A recently published review (Logan and Edwards 2009) indicates that the LightCyclers 1.5 and 2.0 have the capacity to perform the fastest PCR and the Rotor-Gene has the smallest variation in temperature uniformity.
from Logan and Edwards (2009) in Real-Time PCR: Current Technology and Applications
Bibliography:
Peltier devices transfer heat from one side of a semiconductor to another. In general, blocks have significant mass and consequently a degree of thermal inertia. Furthermore, the plastic insulating layer between the reaction vessel and the heater produces an additional thermal lag. As a consequence of this, the temperature transitions are relatively slow and blocks must be very carefully designed to minimise well-to-well variation. Other advances on the Peltier-based technology include its combination with Joule, resistive or convective technology to give improved temperature control and performance across the block.
More recently has been the inclusion of patented Therma-Base technology, whose working principle is based on the evaporation and condensation of a working fluid in a thin vacuum cavity to accurately control well-to-well variation. Three platforms employ alternative heat exchange technologies which permit more rapid thermal ramp rates than blocks, resulting in significantly increased thermocycling speeds. These include a stationary turbulent air-heated glass capillary format (LightCyclers 1.5 and 2.0), a centrifugal air-heated plastic tube format (Rotor-Gene) and a high-thermal-conductivity ceramic heating plate plastic tube format (SmartCycler). For example, the time taken to equilibrate at 72°C using a Rotor-Gene is 0s compared to 15s with a standard 96-well peltier block, resulting in run times that are on average 50% faster. A recently published review (Logan and Edwards 2009) indicates that the LightCyclers 1.5 and 2.0 have the capacity to perform the fastest PCR and the Rotor-Gene has the smallest variation in temperature uniformity.
from Logan and Edwards (2009) in Real-Time PCR: Current Technology and Applications
Bibliography:
- Real-Time PCR: Current Technology and Applications
- Real-Time PCR in Microbiology: From Diagnosis to Characterization
- PCR Troubleshooting: The Essential Guide
- PCR Books
Labels: heating block technology, PCR platform, Therma-Base technology, thermal cycling, thermal engine, thermocycling
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