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Nucleic Acids Res. 2005 Oct 20;33(18):5829-37. Print 2005.

Application of single molecule technology to rapidly map long DNA and study the conformation of stretched DNA.

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  • 1U. S. Genomics, Inc., 12 Gill Street, Suite 4700, Woburn, MA 01801, USA.

Abstract

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.

PMID:
16243782
[PubMed - indexed for MEDLINE]
PMCID:
PMC1266062
Free PMC Article
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