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A single-step competitive binding assay for mapping of single DNA molecules

Nyberg, Lena K.; Persson, Fredrik; Berg, Johan; Bergstrom, Johanna; Fransson, Emelie; Olsson, Linnea; Persson, Moa; Stalnacke, Antti; Wigenius, Jens and Tegenfeldt, Jonas LU , et al. (2012) In Biochemical and Biophysical Research Communications 417(1). p.404-408
Abstract
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... (More)
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. (C) 2011 Elsevier Inc. All rights reserved. (Less)
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publication status
published
subject
keywords
DNA mapping, Nanofluidic. channels, Competitive assay, Single DNA, molecules, Fluorescence microscopy
in
Biochemical and Biophysical Research Communications
volume
417
issue
1
pages
404 - 408
publisher
Elsevier
external identifiers
  • wos:000299491600069
  • scopus:84855783927
ISSN
1090-2104
DOI
10.1016/j.bbrc.2011.11.128
language
English
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yes
id
9cb67a1f-4515-4b9d-b06c-fad9f96e7efe (old id 2419426)
date added to LUP
2012-03-23 10:42:35
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2017-03-05 03:58:10
@article{9cb67a1f-4515-4b9d-b06c-fad9f96e7efe,
  abstract     = {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. (C) 2011 Elsevier Inc. All rights reserved.},
  author       = {Nyberg, Lena K. and Persson, Fredrik and Berg, Johan and Bergstrom, Johanna and Fransson, Emelie and Olsson, Linnea and Persson, Moa and Stalnacke, Antti and Wigenius, Jens and Tegenfeldt, Jonas and Westerlund, Fredrik},
  issn         = {1090-2104},
  keyword      = {DNA mapping,Nanofluidic. channels,Competitive assay,Single DNA,molecules,Fluorescence microscopy},
  language     = {eng},
  number       = {1},
  pages        = {404--408},
  publisher    = {Elsevier},
  series       = {Biochemical and Biophysical Research Communications},
  title        = {A single-step competitive binding assay for mapping of single DNA molecules},
  url          = {http://dx.doi.org/10.1016/j.bbrc.2011.11.128},
  volume       = {417},
  year         = {2012},
}