Title: Reaction-mapped quantitative multiplexed polymerase chain reaction on a microfluidic device
Authors: Ueberfeld, Joern
McKenna, Brian
Rubin-Bejerano, Ifat
Verstrepen, Kevin
Ehrlich, Daniel J # ×
Issue Date: Oct-2008
Publisher: Amer chemical soc
Series Title: Analytical chemistry vol:80 issue:19 pages:7430-7436
Abstract: We have applied multiple-time-point reaction mapping to generate high-dynamic-range quantitative data from PCR multiplexes. The approach measures, then compensates, numerous PCR slope nonidealities across the multiplex without prejudice. A multilane microelectophoresis device with a novel scanning detector that reports redundantly over more than six decades in signal strength was used to collect data with multiple readings for each amplification point and with double internal calibration (lane standards and gene standards). We investigated scaling, properties and sensitivity for readout of 12plex PCR reactions. The sensitive detection, stemming from confocal optics, allowed reduction of the PCR cycle number by approximately five cycles compared to commercial fluorometric readout. This increased sensitivity appears to allow quantitative PCR over a dynamic range of >9 log(2) abundance ratio in multiplex reactions exceeding 20plexes. We argue that the combination of mapping, multiplexing, and an internal standard, improves the per-well efficiency of quantitative expression analysis by a factor of 50-100 relative to fluorometric qPCR readout. Therefore, the approach is attractive for analysis of large gene networks at reduced cost.
ISSN: 0003-2700
Publication status: published
KU Leuven publication type: IT
Appears in Collections:Centre of Microbial and Plant Genetics
× corresponding author
# (joint) last author

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