Diffusion controlled and temperature stable microcapsule reaction compartments for high-throughput microcapsule-PCR

Abstract

A novel approach to perform a high number of individual polymerase chain reactions (PCR) in microcapsule reaction compartments, termed "Microcapsule-PCR" was developed. Temperature stable microcapsules with a selective permeable capsule wall were constructed by matrix-assisted layer-by-layer (LbL) Encapsulation technique. During the PCR, small molecular weight building blocks - nucleotides (dNTPs) were supplied externally and diffuse through the permeable capsule wall into the interior, while the resulted high molecular weight PCR products were accumulated within the microcapsule. Microcapsules (∼110.8 μm average diameter) filled with a PCR reaction mixture were constructed by an emulsion technique having a 2% agarose core and a capsule formed by LbL coating with poly(allylanune-hydrochloride) and poly(4-styrene-sulfonate). An encapsulation efficiency of 47% (measured for primer-FITC (22 bases)) and 98% PCR efficiency was achieved. Microcapsules formed by eight layers of polyelectrolyte and subjected to PCR cycling (up to 95°C) demonstrated good temperature stability without any significantly changes in DNA retention yield and microcapsule morphology. A multiplex Microcapsule-PCR experiment demonstrated that microcapsules are individual compartment and do not exchange templates or primers between microcapsules during PCR cycling. © 2008 WILEY-VCH Verlag GmbH & Co. KGaA.