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A quantitative PCR instrument, also called real-time PCR machine, is an analytical instrument that amplifies and detects
DNA Deoxyribonucleic acid (; DNA) is a polymer composed of two polynucleotide chains that coil around each other to form a double helix. The polymer carries genetic instructions for the development, functioning, growth and reproduction of al ...
. It combines the functions of a
thermal cycler The thermal cycler (also known as a thermocycler, PCR machine or DNA amplifier) is a laboratory apparatus most commonly used to amplify segments of DNA via the polymerase chain reaction (PCR). Thermal cyclers may also be used in laboratories to faci ...
and a
fluorimeter A fluorometer, fluorimeter or fluormeter is a device used to measure parameters of visible spectrum fluorescence: its luminous intensity, intensity and wavelength distribution of emission spectrum after Excitation spectrum, excitation by a cert ...
, enabling the process of
quantitative PCR A real-time polymerase chain reaction (real-time PCR, or qPCR when used quantitatively) is a laboratory technique of molecular biology based on the polymerase chain reaction (PCR). It monitors the amplification of a targeted DNA molecule duri ...
. Quantitative PCR instruments detect fluorescent signals produced during DNA amplification, which correlate with the amount of DNA generated. This allows for precise quantification of specific DNA present in a sample. These instruments are used in many applications, including gene expression analysis, detection of genetic variations,
genotyping Genotyping is the process of determining differences in the genetic make-up (genotype) of an individual by examining the individual's DNA sequence using bioassay, biological assays and comparing it to another individual's sequence or a reference seq ...
, and
diagnostics Diagnosis (: diagnoses) is the identification of the nature and cause of a certain phenomenon. Diagnosis is used in a lot of different disciplines, with variations in the use of logic, analytics, and experience, to determine " cause and effect". ...
of bacterial and viral pathogens. The first quantitative PCR machine was described in 1993, and two commercial models became available in 1996. By 2009, eighteen different models were offered by seven different manufacturers. Prices range from about 4,500 to 150,000 USD. Many configurations of real-time PCR instruments became available on the market, with most commonly used systems designed to accommodate 96- or 384-well plates. Principal performance dimensions include thermal control, fluorescence detection (fluorimetry), and sample throughput. A quantitative PCR instrument is usually equipped with integrated software for real-time data acquisition and analysis, including quantification,
melting curve analysis Melting curve analysis is an assessment of the dissociation characteristics of double-stranded DNA during heating. As the temperature is raised, the double strand begins to dissociate leading to a rise in the absorbance intensity, hyperchromicity. ...
, and quality control metrics. Most systems use Peltier-based thermal blocks.


Thermal control

Efficient performance of quantitative PCR requires rapid, precise, thermal control. 30 cycles of PCR have been demonstrated in less than 10 minutes. Rapid cycling provides several benefits, including, reduced time to result, increased system throughput and improved reaction specificity. In practice however, engineering trade-offs between ease of use, temperature uniformity, and speed, mean that reaction times are typically more than 25 minutes. Thermal non-uniformity during temperature cycling contributes to variability in PCR and, unfortunately, some thermocyclers do not meet the specifications claimed by manufacturers. Increasing the speed of thermal cycling generally reduces thermal uniformity, and can reduce the
precision Precision, precise or precisely may refer to: Arts and media * ''Precision'' (march), the official marching music of the Royal Military College of Canada * "Precision" (song), by Big Sean * ''Precisely'' (sketch), a dramatic sketch by the Eng ...
of
quantitative PCR A real-time polymerase chain reaction (real-time PCR, or qPCR when used quantitatively) is a laboratory technique of molecular biology based on the polymerase chain reaction (PCR). It monitors the amplification of a targeted DNA molecule duri ...
. The temperature uniformity also has a direct effect on the ability to discriminate different PCR products by performing melting point analysis. In addition to uniformity, the resolution with which instruments are able to control temperature is a factor which affects their performance when performing high resolution melting analyses. Therefore, speed, precision and uniformity of thermal control are important performance characteristics of quantitative PCR instruments.


Fluorimetry

Quantitative PCR instruments monitor the progress of PCR, and the nature of amplified products, by measuring
fluorescence Fluorescence is one of two kinds of photoluminescence, the emission of light by a substance that has absorbed light or other electromagnetic radiation. When exposed to ultraviolet radiation, many substances will glow (fluoresce) with colore ...
. The range of different fluorescent labels that can be monitored, the precision with which they can be measured, and the ability to discriminate signals from different labels, are relevant performance characteristics. By using an instrument with sufficient optical channels and extensive assay optimisation, up to 7 separate targets can be simultaneously quantified in a single PCR reaction. However, even with extensive optimisation, the effective dynamic range of such multiplex assays is often reduced due to interference between the constituent reactions. The noise in fluorescence measurements affects the precision of qPCR. It is typically a function of excitation source intensity variation, detector noise and mechanical noise. Multi factorial analysis has suggested that the contribution of mechanical noise is the most important factor, and that systems with no moving parts in their optical paths are likely to provide improved quantitative precision. In addition, when performing high resolution melting analyses, one factor that affects the sensitivity of
heteroduplex A heteroduplex is a double-stranded ( duplex) molecule of nucleic acid originated through the genetic recombination of single complementary strands derived from ''different'' sources, such as from different homologous chromosomes or even from diff ...
detection is fluorimetric precision. Therefore, the number of optical channels and the level of noise in fluorescence measurements are also important performance characteristics of
quantitative PCR A real-time polymerase chain reaction (real-time PCR, or qPCR when used quantitatively) is a laboratory technique of molecular biology based on the polymerase chain reaction (PCR). It monitors the amplification of a targeted DNA molecule duri ...
instruments.


See also

* Real-time PCR


References

{{Reflist


External links


Available real-time PCR cyclers
Molecular biology laboratory equipment Polymerase chain reaction