  An approach for multiplex qualitative and quantitative microarray-based PCR
analysis has been proposed. The characteristics of PCR executed on a gel-based
oligonucleotide microarray with immobilized forward primers and a single common
reverse primer in solution were investigated for several DNA targets. One-stage
multiplex on-chip PCR was studied for simultaneous amplification of herpes
simplex viruses types 1 and 2, cytomegalovirus DNA, and bacteriophage lambda
DNA as an internal control. Additionally the joint analysis of increased number
of targets (with addition of Chlamydia trachomatis, Mycoplasma hominis, and
Ureaplasma urealyticum DNA) was done in two-stage version of assay: first stage
was in-tube PCR with target-specific primers, while the reverse ones contained
5'-adapter region; the second stage was on-chip amplification with immobilized
target-specific forward primers and adapter as common reverse primer in
solution. The possible application of one-stage reaction for human cDNA
analysis was additionally demonstrated with utilization of a common
poly-T-containing primer in solution. SYBR green I; and Cy-5 labeled dUTP were
used for real-time and end-point detection of specific PCR products. The
efficiencies of both one-stage and two-stage reactions was shown to be strongly
dependent on magnesium and primers concentrations. Quantitative PCR in the both
versions was studied with 10-fold serial dilutions of phage lambda DNA. The
method enabled detection of 6 DNA copies per reaction for both versions of
assay. The quantitative interval for one-stage reaction covered eight orders of
concentration. The revealed significant effect of gel pad size on microarray
PCR effectiveness has been discussed.
