PCR Amplification on a Microarray of Gel-Immobilized Oligonucleotides: Detection of Bacterial Toxin- and Drug-Resistant Genes and Their Mutations

Abstract

PCR amplification on a microarray of gel-immobilized primers (microchip) has been developed. One of a pair of PCR primers was immobilized inside a separate microchip polyacrylamide porous gel pad of 0.1 x 0.1 x 0.02 (or 0.04) micron 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 the fluorescently labeled reverse primers, as well as all components of the amplification reaction, diffused into the gel pads from the solution. To increase the amplification efficiency, the forward primers were also added into the solution. The kinetics of amplification was measured in real time in parallel for all gel pads with a fluorescent microscope equipped with a charge-coupled device (CCD) camera. The accuracy of the amplification was assessed by using the melting curves obtained for the duplexes formed by the labeled amplification product and the gel-immobilized primers during the amplification process; alternatively, the duplexes were produced by hybridization 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 identify the Shiga toxin gene and discriminate it from the Shiga-like one. The genomic mutations responsible for rifampicin resistance of the Mycobacterium tuberculosis strains were detected by the same type of PCR amplification of the rpoB gene fragment isolated from sputum of tuberculosis patients. The on-chip PCR amplification has been shown to be a rapid, inexpensive and powerful tool to test genes responsible for bacterial toxin production and drug resistance, as well as to reveal point nucleotide mutations.

DNA microarrays and their applications in medical microbiology

“…Oligonucleotide based DNA arrays have been used for parallel species identification and rifampin resistancerelated mutations in mycobacteria (Troesch et al, 1999) and, more specifically, for the detection of M. tuberculosis strains that are resistant to rifampin (Yue et al, 2004) or isoniazid, kanamycin, streptomycin, pyrazinamide, and ethambutol (Gegia et al, 2008). Oligonucleotide microarrays were developed to analyze and identify drugresistant M. tuberculosis strains, and it was found that the results were comparable with those of standard antimicrobial susceptibility testing (Strizhkov et al, 2000;Mikhailovich et al, 2001). A low-cost and -density DNA microarray was designed to detect mutations that confer isoniazid and rifampin resistance in M. tuberculosis isolates.…”

The role of DNA diffusion in solid phase polymerase chain reaction with gel-immobilized primers in planar and capillary microarray format

“…For real-time measurements of SYBR ® green I fluorescence, the experimental setup that was described earlier consisted of fluorescent microscope equipped with thermotable, a charge coupled device camera, a temperature controller, 13,19,20 and specialized software IMAGEEXPRESS ® ͑Biochip-EIMB, Russia͒. Images were acquired every cycle at the end of elongation step.…”

“…12 An independent cohort of researchers has been working on implementation of SP-PCR to amplification of specific targets on microarray. The suitability of microarray-based SP-PCR for detection of mutations and polymorphisms, [13][14][15][16] detection of toxin genes and microorganisms, 13,[17][18][19][20] eukaryotic expression profiling, 20 methylation analysis, 21 and generation a͒ of extremely long microarray probes 22 has been successfully demonstrated. Some of these works utilized the traditional immobilization of solid phase primers on a functionalized glass surface, while the others used a polyacrylamide gel as a support.…”

“…Some of these works utilized the traditional immobilization of solid phase primers on a functionalized glass surface, while the others used a polyacrylamide gel as a support. 13,14,[18][19][20] The latter approach was particularly successful as the immobilization in a gel separates a primer from solid support 23 facilitating the PCR. 10 Despite that the SP-PCR is believed to have severe limitations compared to traditional PCR, as little as ten copies of microbial DNA were successfully detected and quantified in gelbased SP-PCR assay.…”

Diversity of Human Immunodeficiency Virus Type 1 Subtype A and CRF03_AB Protease in Eastern Europe: Selection of the V77I Variant and Its Rapid Spread in Injecting Drug User Populations

“…To obtain such information, we analyzed HIV-1 genetic variants, along with conventional sequencing, by using oligonucleotide microarrays that have already been proven to be used as a diagnostic tool for identifying nucleic acid polymorphisms (24,29,41).…”