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.