A Simple Microfluidic Assay for the Detection of Ligation Product
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SHORT COMMUNICATION
A Simple Microfluidic Assay for the Detection of Ligation Product Lei Zhang • Jingjing Wang • Johann Roebelen Anubhav Tripathi
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Published online: 22 January 2015 Ó Springer International Publishing Switzerland 2015
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.
L. Zhang, J. Wang and J. Roebelen contributed equally to this work. L. Zhang J. Wang A. Tripathi (&) Center for Biomedical Engineering, School of Engineering, Brown University, 182 Hope Street, Providence, RI 02912, USA e-mail: [email protected] J. Roebelen Feinstein College of Arts and Sciences, Roger Williams University, Bristol, RI, USA
Key Points We designed a novel and rapid microfluidic assay to detect the presence of ligation products. This method can be used to detect point mutations without electrophoresis or melting curve analysis. 1 Introduction Various methods of detecting single nucleotide base-pair mutations exist today [1–4]. A prominent mechanism used to determine the presence of point mutations is through DNA ligation at the position of the mutation [1, 3, 5, 6]. DNA ligases facilitate the joining of nicks on doublestranded DNA duplexes. The optimal rate of nick joining occurs when the sequences around the nick are fully complementary to its duplex strand. When a mismatch occurs, the rate of ligation is severely retarded. By designing oligonucleotide probes that are fully complementary to a variant sequence (mutant [MT]) of interest, the target sequences can be distinguished from other sequences (wild-type [WT]) which differ by only a single nucleotide. Ligation-based point mutation assays include oligonucleotide ligation assay (OLA), ligation chain reaction (LCR) [7], ligation amplification assay (LigAmp) [1, 8], and ligation nucleic acid sequence-based amplification (LNASBA) [9]. OLA relies on a combination of size-based capillary gel electrophoresis separation and different fluorophore-coupled probes to identify target strands from WT strands. LCR and LigAmp both rely on the polymerase chain reaction (PCR) amplification of the ligation products. LNASBA performs isotherma
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