DNA mapping using microfluidic stretching and single-molecule detection of fluorescent site-specific tags.
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.
Chan
Eugene Y
EY
U.S. Genomics, Inc., Woburn, Massachusetts 01801, USA. echan@usgenomics.com
Goncalves
Nuno M
NM
Haeusler
Rebecca A
RA
Hatch
Amie J
AJ
Larson
Jonathan W
JW
Maletta
Anthony M
AM
Yantz
Gregory R
GR
Carstea
Eugene D
ED
Fuchs
Martin
M
Wong
Gordon G
GG
Gullans
Steven R
SR
Gilmanshin
Rudolf
R
D010582:Bacteriophage lambda / Q000235:genetics; D001482:Base Composition / Q000235:genetics; D020029:Base Pairing / Q000235:genetics; D001483:Base Sequence / Q000235:genetics; D002874:Chromosome Mapping / Q000379:methods*; D004247:DNA / Q000737:chemistry / Q000235:genetics*; D015342:DNA Probes / Q000032:analysis / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D005456:Fluorescent Dyes / Q000032:analysis; D044085:Microfluidics / Q000295:instrumentation / Q000379:methods*; D008856:Microscopy, Fluorescence; D009693:Nucleic Acid Hybridization / Q000379:methods; D020135:Peptide Nucleic Acids / Q000032:analysis / Q000235:genetics; D012996:Solutions
D015342:DNA Probes; D004279:DNA, Viral; D005456:Fluorescent Dyes; D020135:Peptide Nucleic Acids; D012996:Solutions; D004247:DNA