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.
channels
DNA mapping
netropsin
restriction maps
dyes
Competitive assay
Nanofluidic. channels
resolution
double-stranded dna
distamycin
Single DNA
complexes
molecules
Fluorescence microscopy
Författare
Lena Nyberg
Chalmers, Kemi- och bioteknik, Fysikalisk kemi
Fredrik Persson
Chalmers, Fysik
Uppsala universitet
J. Berg
Chalmers, Kemi- och bioteknik
Johanna Bergström
Chalmers, Kemi- och bioteknik
Emelie Fransson
Chalmers, Kemi- och bioteknik
Lisbeth Olsson
Industriell bioteknik
Chalmers, Kemi- och bioteknik
M. Persson
Chalmers, Kemi- och bioteknik
Antti Stålnacke
Chalmers, Kemi- och bioteknik
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 1 404-408Styrkeområden
Nanovetenskap och nanoteknik
Livsvetenskaper och teknik (2010-2018)
Ämneskategorier
Biokemi och molekylärbiologi
Kemi
DOI
10.1016/j.bbrc.2011.11.128
PubMed
22166208