Quantitative Polymerase Chain Reaction

The polymerase chain reaction (PCR) has become a useful tool to identify nucleic acids and pathogens because of its ability to amplify specific genes or gene segments directly.  Although traditional PCR with gel-based assessment of amplicons is very sensitive, it is also time-consuming and only semi-quantitative. 

qPCR
                    
Digital PCR
qPCR, employing techniques such as TaqMan, is significantly more quantitative than gel-based methods.  Theoretically, PCR exponentially amplifies nucleic acids, doubling the quantity with each cycle.   The number of amplification cycles required to reach an arbitrarily detection threshold should allow the computation of starting quantity.

However, many factors complicate this calculation, creating uncertainties and inaccuracies.  These factors include:  initial amplification cycles may not be exponential; PCR amplification eventually plateaus after an uncertain number of cycles; PCR amplification efficiency in a sample may be different from that of reference samples; there may be PCR inhibitors in some biological samples; and there may not be enough starting molecules for amplification to the detection threshold.  Therefore the calibration curve for qPCR does not produce a real concentration of substaintial accuracy or precision, and these limitations make qPCR not ideal for measuring one pathogen in a sea of other organisms or a mutation among many wild-type DNAs.
                    
Digital Polymerase Chain Reaction (dPCR) is a refinement of conventional polymerase chain reaction methods that can be used to directly quantify and clonally amplify nucleic acids, including DNA, cDNA and RNA.

Digital PCR overcomes the difficulties of qPCR by transforming unreliable exponential data from conventional PCR to digital signals that simply indicate whether or not amplification has occurred. Digital PCR is achieved by capturing or isolating each individual nucleic acid molecule present in a sample within many separate chambers, zones or regions that are able to localize and concentrate the amplification product to detectable levels. After PCR amplification, a count of chambers, zones or regions containing PCR end-product is a direct measure of the initial nucleic acids quantity.

The capture or isolation of individual nucleic acid molecules may be effected in capillaries, microemulsions, arrays of miniaturized chambers, or on nucleic acid binding surfaces, and detection is technology independent.


DigitalPCR, cPCR, dPCR, dePCR, d-TAQ, NanoArray and PicoArray
are trademarks of Genomic Nanosystems, LLC, a subsidiary of the Cytonix Corporation.
Copyright 1995 -- 2008.  All rights reserved, Genomic Nanosystems, LLC.

Home / Measuring