Diffusion Controlled and Temperature Stable Microcapsule Reaction Compartments for High-Throughput Microcapsule-PCR

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 (similar to 110.8 mu m 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(allylamine-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 degrees 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.