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A single-step competitive binding assay for mapping of single DNA molecules
- Source :
- Biochemical and Biophysical Research Communications, Biochemical and Biophysical Research Communications; Vol 417
- Publication Year :
- 2012
- Publisher :
- Elsevier BV, 2012.
-
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.
- Subjects :
- Biophysics
02 engineering and technology
Computational biology
Biology
Binding, Competitive
Biochemistry
Fluorescence
03 medical and health sciences
chemistry.chemical_compound
Restriction map
Optical mapping
A-DNA
Molecular Biology
Fluorescent Dyes
030304 developmental biology
Base Composition
Benzoxazoles
0303 health sciences
Quinolinium Compounds
Netropsin
DNA
Cell Biology
Microfluidic Analytical Techniques
Circular permutation in proteins
021001 nanoscience & nanotechnology
Molecular biology
genomic DNA
chemistry
0210 nano-technology
Single molecule real time sequencing
Subjects
Details
- ISSN :
- 0006291X
- Volume :
- 417
- Database :
- OpenAIRE
- Journal :
- Biochemical and Biophysical Research Communications
- Accession number :
- edsair.doi.dedup.....14426dab298999c9d79cfe76c1cd654c