DNA analysis with multiplex microarray-enhanced PCR

Abstract

We have developed a highly sensitive method for DNA analysis on 3D gel element microarrays, a technique we call multiplex microarray-enhanced PCR (MME-PCR). Two amplification strategies are carried out simultaneously in the reaction chamber: on or within gel elements, and in bulk solution over the gel element array. MME-PCR is initiated by multiple complex primers containing gene-specific, forward and reverse, sequences appended to the 3′ end of a universal amplification primer. The complex primer pair is covalently tethered through its 5′ end to the polyacryl- amide backbone. In the bulk solution above the gel element array, a single pair of unattached universal primers simultaneously directs pseudo-monoplex PCR of all targets according to normal solution-phase PCR. The presence of a single universal PCR primer pair in solution accelerates amplification within gel elements and eliminates the problem of primer interference that is common to conventional multiplex PCR. We show 106-fold amplification of targeted DNA after 50 cycles with average amplification efficiency 1.34 per cycle, and demonstrate specific on-chip amplification of six genes in Bacillus subtilis. All six genes were detected at 4.5 pg of bacterial genomic DNA (equivalent to 103 genomes) in 60 independent amplification reactions performed simultaneously in single reaction chamber.

Tween-modified suspension array for sensitive biomolecular detection

“…In order to get a lower limit of detection, many methods have been developed, including three-dimensional (3D) matrixes modification, such as Noble PA group and Bavykin S group developed ployacrylamide for surface modification [11,12], and Gershwin ME group and Dufva M group used agarose for surface coated and polymer [13,14], which could increase the efficiency of biomolecules immobilization. And some groups chose signal amplification technology [15][16][17][18][19], or used more sensitive signal detection and collection apparatus for signal collection.…”

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

“…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.…”

MegaPlex PCR: a strategy for multiplex amplification

“…Several previously developed methods use surface immobilization of primer pairs as a way to separate reactions and thus limit primerdimer formation [4][5][6] , but drawbacks of these methods include inefficient amplification, loss of reactants from the surface and considerable primer-dimer formation within pairs of primers.…”

“…MegaPlex PCR likewise uses physical separation of primer pairs to prevent their interaction, but it additionally includes 'common sequences' at the 5′ ends of the surface primers so that early-stage amplicons can be moved into the solution phase for co-amplification by highly efficient and unbiased PCR using a single common primer pair [6][7][8] (Fig. 1).…”