Single molecule DNA intercalation in continuous homogenous elongational flow

Abstract

Sequence-nonspecific staining of DNA with intercalating fluorophores is required for fluorescence-based length estimation of elongated DNA in optical mapping techniques. However, the observed length of a DNA molecule is affected by the relative concentrations of DNA and dye. In some applications, predetermination of DNA concentration may not be possible. Here we present a microfluidic approach in which individual DNA molecules are entrained by converging laminar sheath flows containing the intercalating dye PO-PRO-1. This provides uniform staining regardless of DNA concentration, and uniform elastic stretching of DNA in continuous elongational flow. On-chip intercalation provides a unique process for concentration-independent staining of long DNA fragments for the optical mapping method Genome Sequence Scanning (GSS), and normalizes intramolecular elasticity across a broad range of molecule lengths. These advances permit accurate mapping of observed molecules to sequence derived templates, thus improving detection of complex bacterial mixtures using GSS.

Analysis of single nucleic acid molecules in micro- and nano-fluidics

“…This method, termed GSS, Genome Sequence Scanning (previously DLA, Direct Linear Analysis) generates a fluorescent barcode along a single DNA molecule that is read as it flows through the multicolor confocal detection region 39 . Improved consistency in both intercalation dye labeling and DNA stretching efficiency was achieved by performing DNA intercalation staining on the device 122 . The device was further optimized to increase throughput and stretching consistency by redesigning the funnel geometry to maintain constant shear through the detection region 123 .…”

“…39 Improved consistency in both intercalation dye labeling and DNA stretching efficiency was achieved by performing DNA intercalation staining on the device. 120 The device was further optimized to increase throughput and stretching consistency by redesigning the funnel geometry to maintain constant shear through the detection region. 121 The platform has been used for bacterial genotyping 122 and strain typing.…”