title: Application of single molecule technology to rapidly map long DNA and study the conformation of stretched DNA.
abstract: Herein we describe the first application of direct linear analysis (DLA) to the mapping of a bacterial artificial chromosome (BAC), specifically the 185.1 kb-long BAC 12M9. DLA is a single molecule mapping technology, based on microfluidic elongation and interrogation of individual DNA molecules, sequence-specifically tagged with bisPNAs. A DNA map with S/N ratio sufficiently high to detect all major binding sites was obtained using only 200 molecule traces. A new method was developed to extract an oriented map from an averaged map that included a mixture of head-first and tail-first DNA traces. In addition, we applied DLA to study the conformation and tagging of highly stretched DNA. Optimal conditions for promoting sequence-specific binding of bisPNA to an 8 bp target site were elucidated using DLA, which proved superior to electromobility shift assays. DLA was highly reproducible with a hybridized tag position localized with an accuracy of +/-0.7 microm or +/-2.1 kb demonstrating its utility for rapid mapping of large DNA at the single molecule level. Within this accuracy, DNA molecules, stretched to at least 85% of their contour length, were stretched uniformly, so that the map expressed in relative coordinates, was the same regardless of the molecule extension.
authors.affiliation: U. S. Genomics, Inc., 12 Gill Street, Suite 4700, Woburn, MA 01801, USA.
authors.forename: Kevin M
authors.initials: KM
authors.lastname: Phillips
authors.forename: Jonathan W
authors.initials: JW
authors.lastname: Larson
authors.forename: Gregory R
authors.initials: GR
authors.lastname: Yantz
authors.forename: Christina M
authors.initials: CM
authors.lastname: D'Antoni
authors.forename: Michael V
authors.initials: MV
authors.lastname: Gallo
authors.forename: Kimberly A
authors.initials: KA
authors.lastname: Gillis
authors.forename: Nuno M
authors.initials: NM
authors.lastname: Goncalves
authors.forename: Lori A
authors.initials: LA
authors.lastname: Neely
authors.forename: Steven R
authors.initials: SR
authors.lastname: Gullans
authors.forename: Rudolf
authors.initials: R
authors.lastname: Gilmanshin
mesh_terms: D002874:Chromosome Mapping / Q000379:methods*; D022202:Chromosomes, Artificial, Bacterial; D004247:DNA / Q000737:chemistry*; D005456:Fluorescent Dyes; D023281:Genomics / Q000379:methods*; D006801:Humans; D046210:Microfluidic Analytical Techniques; D009690:Nucleic Acid Conformation; D015203:Reproducibility of Results; D016324:Sequence Tagged Sites
chemical_list: D005456:Fluorescent Dyes; D004247:DNA