A Simple Microfluidic Assay for the Detection of Ligation Product

Abstract

We present a novel microfluidic-based approach to detect ligation products. The conformal specificity of ligases is used in various molecular assays to detect point mutations. Traditional methods of detecting ligation products include denaturing gel electrophoresis, sequence amplification, and melting curve analysis. Gel electrophoresis is a labor- and time-intensive process, while sequence amplification and melting curve analysis require instruments capable of accurate thermal ramping and sensitive optical detection. Microfluidics has been widely applied in genomics, proteomics, and cell cytometry to enable rapid and automated assays. We designed an assay that fluorogenically detects ligation products following a simple magnetic separation through a microfluidic channel. 100 nM of synthetic HIV-1 K103N minority mutant templates were successfully detected in 30 min. This simple and rapid method can be coupled with any ligation assay for the detection of ligation products.

Advances in Continuous Microfluidics-Based Technologies for the Study of HIV Infection

“…Compared to conventional methods such as gel electrophoresis and melting curve analysis, the use of microfluidics provides compelling advantages in the detection of these mutations. As shown by an in vitro assay developed to discriminate the well-known synthetic K103N point mutation from wild-type template, the technique brings rapidity (30 min), small volumes (5 µL), specificity, and sensitivity in the nanomolar range (100 nM) [77]. The assay includes ligation of specific fluorescent probes to target sequences, duplex denaturation, magnetic microfluidic separation, and fluorogenic detection.…”