DNA Mapping Using Microfluidic Stretching and Single-molecule Detection of Fluorescent Site-specific Tags

Overview

Journal Genome Res

Specialty Genetics

Date 2004 Jun 3

PMID 15173119

Citations 44

Authors

Eugene Y Chan

Nuno M Goncalves

Rebecca A Haeusler

Amie J Hatch

Jonathan W Larson

Anthony M Maletta

Gregory R Yantz

Eugene D Carstea

Martin Fuchs

Gordon G Wong

Steven R Gullans

Rudolf Gilmanshin

Affiliations

Soon will be listed here.

Abstract

We have developed a rapid molecular mapping technology--Direct Linear Analysis (DLA)--on the basis of the analysis of individual DNA molecules bound with sequence-specific fluorescent tags. The apparatus includes a microfluidic device for stretching DNA molecules in elongational flow that is coupled to a multicolor detection system capable of single-fluorophore sensitivity. Double-stranded DNA molecules were tagged at sequence-specific motif sites with fluorescent bisPNA (Peptide Nucleic Acid) tags. The DNA molecules were then stretched in the microfluidic device and driven in a flow stream past confocal fluorescence detectors. DLA provided the spatial locations of multiple specific sequence motifs along individual DNA molecules, and thousands of individual molecules could be analyzed per minute. We validated this technology using the 48.5 kb lambda phage genome with different 8-base and 7-base sequence motif tags. The distance between the sequence motifs was determined with an accuracy of +/-0.8 kb, and these tags could be localized on the DNA with an accuracy of +/-2 kb. Thus, DLA is a rapid mapping technology, suitable for analysis of long DNA molecules.

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