A single-step competitive binding assay for mapping of single DNA molecules
Artikel i vetenskaplig tidskrift, 2012

Optical mapping of genomic DNA is of relevance for a plethora of applications such as scaffolding for sequencing and detection of structural variations as well as identification cif pathogens like bacteria and viruses. For future clinical applications it is desirable to have a fast and robust mapping method based on as few steps as possible. We here demonstrate a single-step method to obtain a DNA barcode that is directly visualized using nanofluidic devices and fluorescence microscopy. Using a mixture of YOYO-1, a bright DNA dye, and netropsin, a natural antibiotic with very high AT specificity, we obtain a DNA map with a fluorescence intensity profile along the DNA that reflects the underlying sequence. The netropsin binds to AT-tetrads and blocks these binding sites from YOYO-1 binding which results in lower fluorescence intensity from AT-rich regions of the DNA. We thus obtain a DNA barcode that is dark in AT-rich regions and bright in GC-rich regions with kilobasepair resolution. We demonstrate the versatility of the method by obtaining a barcode on DNA from the phage T4 that captures its circular permutation and agrees well with its known sequence.

dyes

channels

resolution

Competitive assay

Fluorescence microscopy

molecules

distamycin

DNA mapping

restriction maps

netropsin

double-stranded dna

complexes

Single DNA

Nanofluidic. channels

Författare

Lena Nyberg

Chalmers, Kemi- och bioteknik, Fysikalisk kemi

Fredrik Persson

Göteborgs universitet

J. Berg

Chalmers University of Technology

Johanna Bergström

Chalmers, Kemi- och bioteknik

Emelie Fransson

Chalmers, Kemi- och bioteknik

L. Olsson

Chalmers University of Technology

M. Persson

Chalmers University of Technology

A. Stalnacke

Chalmers University of Technology

Jens Wigenius

Chalmers, Kemi- och bioteknik, Fysikalisk kemi

J. O. Tegenfeldt

Göteborgs universitet

Fredrik Westerlund

Chalmers, Kemi- och bioteknik, Fysikalisk kemi

Biochemical and Biophysical Research Communications

0006-291X (ISSN) 1090-2104 (eISSN)

Vol. 417 404-408

Styrkeområden

Nanovetenskap och nanoteknik

Livsvetenskaper och teknik

Ämneskategorier

Biokemi och molekylärbiologi

Kemi

DOI

10.1016/j.bbrc.2011.11.128

PubMed

22166208