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Mathematical analysis of the real time array PCR (RTA PCR) process

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These file attachments have been under embargo and were made available to the public after the embargo was lifted on 21 June 2012.

Author: Dijksman J.F. · Pierik, A.
Type:article
Date:2012-06-21
Embargo lifted:2012-06-21
Publisher: Elsevier
Institution: Philips Research
Source:Chemical Engineering Science; authors version
Identifier: MS 32.614
Keywords: microarrays · pcr · real time array pcr · real-time pcr
Rights: (c) Elsevier

Abstract

Real Time Array PCR is a recently developed biochemical technique that measures amplification curves (like quantitative real time Polymerase Chain Reaction (qPCR)) of a multitude of different templates ina sample. It combines two different techniques to profit from theadvantages of both techniques, namely qPCR (real time quantitative detection) with microarrays (high multiplex capability). This enablesthe quantitative detection of many more target sequences than can be done by qPCR. Thereby, the concentration of the many different target molecules originally present in a sample can be measured. Labeled primers are used that are first elongated to form labeled amplicons in the bulk and these can hybridize to capture probes immobilizedon the surface of the microarray. During each PCR cycle, there is atime window available during which the formed labeled amplicons canhybridize to the target sequences on the microarray surface. By detection of the fluorescence of the spots on the microarray, amplification curves comparable to real time PCR can be obtained, which can be used to deduce the information needed on the presence and the amount of targets originally present in the sample. We present a mathematical model that provides fundamental insights in the different steps of Real Time Array PCR and that can be used to optimize the different biochemical processes taking place. At the microarray surface specific molecules are captured and taken away from the solution, causing a concentration gradient that powers a material flow towards themicroarray surface. Only the labeled strand of the amplicon is captured by the probes on the microarray surface and as a result locallythe PCR process is not symmetric anymore. Moreover, in course of the process more and more ssDNA renatures, leaving relatively less strands and complexes available for hybridization. We found that to a large extent, however, the surface fluorescence scales with the bulkconcentration. Important parameters to optimize are the enzyme concentration and degradation, the primer concentration and the capture probe decay rate. Also the surface hybridization time is critical since the time to reach a steady state is at least one order of magnitude longer compared to the timing of the bulk processes in qPCR.

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