title: 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.
authors.affiliation: PathoGenetix, Woburn, MA 01801, USA. robmeltzer1@gmail.com.
authors.forename: Joshua W
authors.initials: JW
authors.lastname: Griffis
authors.forename: Mikhail M
authors.initials: MM
authors.lastname: Safranovitch
authors.forename: Shilpi P
authors.initials: SP
authors.lastname: Vyas
authors.forename: Sean
authors.initials: S
authors.lastname: Gerrin
authors.forename: Ekaterina
authors.initials: E
authors.lastname: Protozanova
authors.forename: Gene
authors.initials: G
authors.lastname: Malkin
authors.forename: Robert H
authors.initials: RH
authors.lastname: Meltzer
mesh_terms: D001583:Benzoxazoles / Q000737:chemistry; D004247:DNA / Q000032:analysis* / Q000737:chemistry*; D004548:Elasticity; D005456:Fluorescent Dyes / Q000737:chemistry; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation* / Q000379:methods
chemical_list: D001583:Benzoxazoles; D005456:Fluorescent Dyes; C514113:Po-Pro-1; D004247:DNA