Single-molecule denaturation mapping of DNA in nanofluidic channels.
Here we explore the potential power of denaturation mapping as a single-molecule technique. By partially denaturing YOYO-1-labeled DNA in nanofluidic channels with a combination of formamide and local heating, we obtain a sequence-dependent "barcode" corresponding to a series of local dips and peaks in the intensity trace along the extended molecule. We demonstrate that this structure arises from the physics of local denaturation: statistical mechanical calculations of sequence-dependent melting probability can predict the barcode to be observed experimentally for a given sequence. Consequently, the technique is sensitive to sequence variation without requiring enzymatic labeling or a restriction step. This technique may serve as the basis for a new mapping technology ideally suited for investigating the long-range structure of entire genomes extracted from single cells.
Reisner
Walter
W
Department of Physics, McGill University, Montreal, QC, Canada. reisner@physics.mcgill.ca
Larsen
Niels B
NB
Silahtaroglu
Asli
A
Kristensen
Anders
A
Tommerup
Niels
N
Tegenfeldt
Jonas O
JO
Flyvbjerg
Henrik
H
D000465:Algorithms; D001435:Bacteriophages / Q000235:genetics; D001583:Benzoxazoles / Q000737:chemistry; D004247:DNA / Q000737:chemistry* / Q000235:genetics; D005559:Formamides / Q000737:chemistry; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation / Q000379:methods*; D008956:Models, Chemical; D036103:Nanotechnology / Q000295:instrumentation / Q000379:methods*; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D011806:Quinolinium Compounds / Q000737:chemistry; D044366:Transition Temperature
D001583:Benzoxazoles; D005559:Formamides; D011806:Quinolinium Compounds; C075296:1,1'-((4,4,7,7-tetramethyl)-4,7-diazaundecamethylene)bis-4-(3-methyl-2,3-dihydro(benzo-1,3-oxazole)-2-methylidene)quinolinium; C031066:formamide; D004247:DNA