Microfluidic Reactor for Sequential Operation of Polymerase Chain Reaction/Ligase Detection Reaction

Masahiko Hashimoto, Kazuhik

Abstract

We have developed a microfluidic bioreactor for detecting single base mutations in genomic DNA, where a primary polymerase chain reaction (PCR) and an allelespecific ligation detection reaction (LDR) were conducted in sequence. The effect of carryover from the primary PCR on the subsequent LDR was thoroughly investigated in terms of LDR yield and fidelity, and we found that a post-PCR treatment for the amplicons prior to its incorporation into the LDR phase was not necessarily required, which allowed us to use a simple diffusive mixer to online mix the post- PCR solution with the LDR reagents. We also performed a numerical analysis to roughly estimate a channel length required for the diffusive mixing. We successfully demonstrated the ability of the system to detect one mutant DNA in 1000 normal sequences at a relatively high processing speed (total processing time = ca. 28.1 min).