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An approach for multiplex qualitative and quantitative microarray-based PCRanalysis has been proposed. The characteristics of PCR executed on a gel-basedoligonucleotide microarray with immobilized forward primers and a single commonreverse primer in solution were investigated for several DNA targets. One-stagemultiplex on-chip PCR was studied for simultaneous amplification of herpessimplex viruses types 1 and 2, cytomegalovirus DNA, and bacteriophage lambdaDNA as an internal control. Additionally the joint analysis of increased numberof targets (with addition of Chlamydia trachomatis, Mycoplasma hominis, andUreaplasma urealyticum DNA) was done in two-stage version of assay: first stagewas in-tube PCR with target-specific primers, while the reverse ones contained5'-adapter region; the second stage was on-chip amplification with immobilizedtarget-specific forward primers and adapter as common reverse primer insolution. The possible application of one-stage reaction for human cDNAanalysis was additionally demonstrated with utilization of a commonpoly-T-containing primer in solution. SYBR green I; and Cy-5 labeled dUTP wereused for real-time and end-point detection of specific PCR products. Theefficiencies of both one-stage and two-stage reactions was shown to be stronglydependent on magnesium and primers concentrations. Quantitative PCR in the bothversions was studied with 10-fold serial dilutions of phage lambda DNA. Themethod enabled detection of 6 DNA copies per reaction for both versions ofassay. The quantitative interval for one-stage reaction covered eight orders ofconcentration. The revealed significant effect of gel pad size on microarrayPCR effectiveness has been discussed.

Simultaneous Qualitative And Quantitative Identification Of Multiple Targets By Polymerase Chain Reaction On Microarray