Reverse transcription-polymerase chain reaction on a microarray: the integrating concept of "active arrays".

In this report we describe the proof of principle of a reverse transcription polymerase chain reaction (RT-PCR) but on-chip, with immobilized specific primers using a transcriptome of mouse-muscle fibroblasts for detection of muscle-specific expression products of these cells. The isolated total mRNA was directly incubated on an array of immobilized and solubilized specific primers, which allow the amplification of certain muscle-specific RNAs via its immobilized cDNAs. In contrast to others, the immobilized cDNA-products were directly synthesized on the chip by applying covalently bound specific primers. The products were detected by the incorporated and fluorophore-modified specific primers of the subsequently synthezised second strand. In addition, this second-strand served as a further template (like the basically used mRNA) in the subsequent solid-phase-PCR to amplify first-strand cDNA copies at the remaining immobilized specific primer-probes. This is the intrinsic factor of the amplification of certain signals of this application. The specific cDNA templates of genes coding for subunits of the mouse muscle acetylcholine receptor (Chrna1, Chrnb1, Chrnd) and the genes coding for myogenin (Myog), muscle creatine kinase (Ckmm), and ATPase (Atp2a2) were amplified on a biochip by RT-PCR directly from freshly isolated mRNA. The resulting procedure allows the detection of mRNA sequences from less than 5 pg of total RNA preparations.

On-chip PCR amplification of very long templates using immobilized primers on glassy surfaces.

In this paper we describe a novel method for visualizing very long DNA fragments (for example >6 kb) which are difficult to spot with commonly used arrayers or capillary samplers with very small nanoliter volumes, using directly bound primers on "on-chip" polymerase chain reaction (PCR). We have used the genomes of the M13 bacteriophage (7.2 kb) the human mitochondrion (16.5 kb) as examples of long DNA templates to test the PCR and were able to elicit robust reactivity. Over 75% of the immobilized primers could be elongated to their fullest extent. In addition we were able to elicit the PCR reaction with double stranded templates in which one primer was immobilized and the other suspended in the reaction solution. These synthesized PCR products were visualized by either confocal microarray scanning or fluorescence microscopy using Cy5-dye fluorescence of the modified free primer, or the fluorescence of intercalating dyes.