Work

Fluorescent Marker for Direct Detection of Specific dsDNA Sequences

Year: 2009

Type: article

Abstract: We have created a fluorescent marker using a mutant EcoRI restriction endonuclease (K249C) that enables prolonged, direct visualization of specific sequences on genomic lengths of double-stranded (ds) DNA. The marker consists of a biotinylated enzyme, attached through the biotin-avidin interaction to a fluorescent nanosphere. Control over biotin position with respect to the enzyme's binding pocket is achieved by biotinylating the mutant EcoRI at the mutation site. Biotinylated enzyme is incubated with dsDNA and NeutrAvidin-coated, fluorescent nanospheres under conditions that allow enzyme binding but prevent cleavage. Marker-laden DNA is then fluorescently stained and stretched on polylysine-coated glass slides so that the positions of the bound markers along individual DNA molecules can be measured. We demonstrate the marker's ability to bind specifically to its target sequence using both bulk gel-shift assays and single-molecule methods.

Source: Analytical Chemistry

DOI: 10.1021/ac9019895

Authors: Rebecca Dylla-Spears, Jacqueline E. Townsend, Lydia L. Sohn, Linda Jen-Jacobson, Susan J. Muller

Institutions: University of Pittsburgh, University of California, Berkeley

Language: English

Topic: Advanced biosensing and bioanalysis techniques

Subfield: Molecular Biology

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

Open Access status: green

Funders: National Human Genome Research Institute, National Institute of General Medical Sciences

Awards: Sequence Specific DNA Protein Interactions, Single Molecule Genotyping Using Microfluidic Stagnation Point Flows, Sequence specific DNA protein interactions