Application of single molecule technology to rapidly map long DNA and study the conformation of stretched DNA
| Autor: | Kimberly A. Gillis, Michael Gallo, Gregory R. Yantz, Steven R. Gullans, Nuno Goncalves, Christina M. D’Antoni, Rudolf Gilmanshin, Kevin M. Phillips, Lori Anne Neely, Jonathan W. Larson |
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| Rok vydání: | 2005 |
| Předmět: |
Chromosomes
Artificial Bacterial Microfluidics 02 engineering and technology Biology Article 03 medical and health sciences chemistry.chemical_compound Genetics Humans Molecule A-DNA Binding site Fluorescent Dyes Sequence Tagged Sites 030304 developmental biology 0303 health sciences Bacterial artificial chromosome Chromosome Mapping Reproducibility of Results DNA Genomics Microfluidic Analytical Techniques 021001 nanoscience & nanotechnology Molecular biology chemistry Biophysics Nucleic Acid Conformation Elongation 0210 nano-technology Single molecule real time sequencing |
| Zdroj: | Nucleic Acids Research |
| ISSN: | 1362-4962 0305-1048 |
| DOI: | 10.1093/nar/gki895 |
| Popis: | Herein we describe the first application of direct linear analysis (DLA) to the mapping of a bacterial artificial chromosome (BAC), specifically the 185.1 kb-long BAC 12M9. DLA is a single molecule mapping technology, based on microfluidic elongation and interrogation of individual DNA molecules, sequence-specifically tagged with bisPNAs. A DNA map with S/N ratio sufficiently high to detect all major binding sites was obtained using only 200 molecule traces. A new method was developed to extract an oriented map from an averaged map that included a mixture of head-first and tail-first DNA traces. In addition, we applied DLA to study the conformation and tagging of highly stretched DNA. Optimal conditions for promoting sequence-specific binding of bisPNA to an 8 bp target site were elucidated using DLA, which proved superior to electromobility shift assays. DLA was highly reproducible with a hybridized tag position localized with an accuracy of +/-0.7 microm or +/-2.1 kb demonstrating its utility for rapid mapping of large DNA at the single molecule level. Within this accuracy, DNA molecules, stretched to at least 85% of their contour length, were stretched uniformly, so that the map expressed in relative coordinates, was the same regardless of the molecule extension. |
| Databáze: | OpenAIRE |
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