title: Rapid DNA mapping by fluorescent single molecule detection.
abstract: DNA mapping is an important analytical tool in genomic sequencing, medical diagnostics and pathogen identification. Here we report an optical DNA mapping strategy based on direct imaging of individual DNA molecules and localization of multiple sequence motifs on the molecules. Individual genomic DNA molecules were labeled with fluorescent dyes at specific sequence motifs by the action of nicking endonuclease followed by the incorporation of dye terminators with DNA polymerase. The labeled DNA molecules were then stretched into linear form on a modified glass surface and imaged using total internal reflection fluorescence (TIRF) microscopy. By determining the positions of the fluorescent labels with respect to the DNA backbone, the distribution of the sequence motif recognized by the nicking endonuclease can be established with good accuracy, in a manner similar to reading a barcode. With this approach, we constructed a specific sequence motif map of lambda-DNA. We further demonstrated the capability of this approach to rapidly type a human adenovirus and several strains of human rhinovirus.
authors.affiliation: Cardiovascular Research Institute and Center for Human Genetics, University of California San Francisco, CA 94115, USA.
authors.forename: Ming
authors.initials: M
authors.lastname: Xiao
authors.forename: Angie
authors.initials: A
authors.lastname: Phong
authors.forename: Connie
authors.initials: C
authors.lastname: Ha
authors.forename: Ting-Fung
authors.initials: TF
authors.lastname: Chan
authors.forename: Dongmei
authors.initials: D
authors.lastname: Cai
authors.forename: Lucinda
authors.initials: L
authors.lastname: Leung
authors.forename: Eunice
authors.initials: E
authors.lastname: Wan
authors.forename: Amy L
authors.initials: AL
authors.lastname: Kistler
authors.forename: Joseph L
authors.initials: JL
authors.lastname: DeRisi
authors.forename: Paul R
authors.initials: PR
authors.lastname: Selvin
authors.forename: Pui-Yan
authors.initials: PY
authors.lastname: Kwok
mesh_terms: D000260:Adenoviruses, Human / Q000145:classification / Q000235:genetics; D010582:Bacteriophage lambda / Q000235:genetics; D004247:DNA / Q000737:chemistry; D004279:DNA, Viral / Q000737:chemistry; D004706:Endodeoxyribonucleases; D005456:Fluorescent Dyes / Q000032:analysis*; D016679:Genome, Viral; D023281:Genomics / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods*; D012229:Rhinovirus / Q000145:classification / Q000235:genetics
chemical_list: D004279:DNA, Viral; D005456:Fluorescent Dyes; D004247:DNA; D004706:Endodeoxyribonucleases