PCR amplification on a microarray of gel-immobilized oligonucleotides: Detection of bacterial toxin- and drug-resistant genes and their mutations

PCR amplification on a microarray of gel-immobilized primers (microchip) ha s been developed. One of a pair of PCR primers was immobilized inside a sep arate microchip polyacrylamide porous gel pad of 0.1 x 0.1 x 0.02 (or 0.04) mu m in size and 0.2 (or 0.4) nL in volume. The amplification was carried out simultaneously both in solution covering the microchip array and inside gel pads. Each gel pad contained the immobilized forward primers, while th e fluorescently labeled reverse primers, as well as all components of the a mplification reaction, diffused into the gel pads from the solution. To inc rease the amplification efficiency the forward primers were also added into the solution. The kinetics of amplification was measured in real time in p arallel for all gel pads with a fluorescent microscope equipped with a char ge-coupled device (CCD) camera. The accuracy of the amplification was asses sed by using the melting curves obtained for the duplexes formed by the lab eled amplification product and the gel-immobilized primers during the ampli fication process; alternatively, the duplexes were produced by hybridizatio n of the extended immobilized primers with labeled oligonucleotide probes. The on-chip amplification was applied to detect the anthrax toxin genes and the plasmid-borne beta-lactamase gene responsible for bacterial ampicillin resistance. The allele-specific type of PCR amplification was used to iden tify the Shiga toxin gene and discriminate it from the Shiga-like one. The genomic mutations responsible for rifampicin resistance of the Mycobacteriu m tuberculosis strains were detected by the same type of PCR amplification of the rpoB gene fragment isolated from sputum of tuberculosis patients. Th e on-chip PCR amplification has been shown to be a rapid, inexpensive and p owerful tool to test genes responsible for bacterial toxin production and d rug resistance, as well as to reveal point nucleotide mutations.