Application of single molecule technology to rapidly map long DNA and study the conformation of stretched DNA.
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.
Phillips
Kevin M
KM
U. S. Genomics, Inc., 12 Gill Street, Suite 4700, Woburn, MA 01801, USA.
Larson
Jonathan W
JW
Yantz
Gregory R
GR
D'Antoni
Christina M
CM
Gallo
Michael V
MV
Gillis
Kimberly A
KA
Goncalves
Nuno M
NM
Neely
Lori A
LA
Gullans
Steven R
SR
Gilmanshin
Rudolf
R
D002874:Chromosome Mapping / Q000379:methods*; D022202:Chromosomes, Artificial, Bacterial; D004247:DNA / Q000737:chemistry*; D005456:Fluorescent Dyes; D023281:Genomics / Q000379:methods*; D006801:Humans; D046210:Microfluidic Analytical Techniques; D009690:Nucleic Acid Conformation; D015203:Reproducibility of Results; D016324:Sequence Tagged Sites
D005456:Fluorescent Dyes; D004247:DNA