Real time quantitative amplification detection on a microarray: towards high multiplex quantitative PCR

Lab Chip. 2012 Apr 24;12(10):1897-902. doi: 10.1039/c2lc20740k. Epub 2012 Apr 3.

Abstract

Quantitative real-time polymerase chain reaction (qrtPCR) is widely used as a research and diagnostic tool. Notwithstanding its many powerful features, the method is limited in the degree of multiplexing to about 6 due to spectral overlap of the available fluorophores. A new method is presented that allows quantitative amplification detection at higher multiplexing by the integration of amplification in solution and monitoring via hybridization to a microarray in real-time. This method does not require any manipulation of the PCR product and runs in a single closed chamber. Employing labeled primers, one of the main challenges is to measure surface signals against a high fluorescence background from solution. A compact, confocal scanner is employed, based on miniaturized optics from DVD technology and combined with a flat thermocycler for simultaneous scanning and heating. The feasibility of this method is demonstrated in singleplex with an analytical sensitivity comparable to routine qrtPCR.

MeSH terms

  • Bacterial Proteins / genetics
  • DNA Primers
  • Equipment Design
  • Fluorescent Dyes
  • Models, Genetic
  • Oligonucleotide Array Sequence Analysis / instrumentation*
  • Oligonucleotide Array Sequence Analysis / methods*
  • Real-Time Polymerase Chain Reaction / instrumentation*
  • Real-Time Polymerase Chain Reaction / methods*
  • Sensitivity and Specificity
  • Spectrometry, Fluorescence

Substances

  • Bacterial Proteins
  • DNA Primers
  • FemA protein, Bacteria
  • Fluorescent Dyes