Single DNA molecule stretching in sudden mixed shear and elongational microflows.
High-throughput stretching and monitoring of single DNA molecules in continuous elongational flow offers compelling advantages for biotechnology applications such as DNA mapping. However, the polymer dynamics in common microfluidic implementations are typically complicated by shear interactions. These effects were investigated by observation of fluorescently labeled 185 kb bacterial artificial chromosomes in sudden mixed shear and elongational microflows generated in funneled microfluidic channels. The extension of individual free DNA molecules was studied as a function of accumulated fluid strain and strain rate. Under constant or gradually changing strain rate conditions, stretching by the sudden elongational component proceeded as previously described for an ideal elongational flow (T. T. Perkins, D. E. Smith and S. Chu, Science, 1997, 276, 2016): first, increased accumulated fluid strain and increased strain rate produced higher stretching efficiencies, despite the complications of shear interactions; and second, the results were consistent with unstretched molecules predominantly in hairpin conformations. More abrupt strain rate profiles did not deliver a uniform population of highly extended molecules, highlighting the importance of balance between shear and elongational components in the microfluidic environment for DNA stretching applications. DNA sizing with up to 10% resolution was demonstrated. Overall, the device delivered 1000 stretched DNA molecules per minute in a method compatible with diffraction-limited optical sequence motif mapping and without requiring laborious chemical modifications of the DNA or the chip surface. Thus, the method is especially well suited for genetic characterization of DNA mixtures such as in pathogen fingerprinting amidst high levels of background DNA.
Larson
Jonathan W
JW
US Genomics, Inc., Woburn, MA 01801, USA.
Yantz
Gregory R
GR
Zhong
Qun
Q
Charnas
Rebecca
R
D'Antoni
Christina M
CM
Gallo
Michael V
MV
Gillis
Kimberly A
KA
Neely
Lori A
LA
Phillips
Kevin M
KM
Wong
Gordon G
GG
Gullans
Steven R
SR
Gilmanshin
Rudolf
R
D010582:Bacteriophage lambda / Q000235:genetics; D001583:Benzoxazoles / Q000737:chemistry; D022202:Chromosomes, Artificial, Bacterial / Q000737:chemistry; D015342:DNA Probes / Q000737:chemistry; D004279:DNA, Viral / Q000737:chemistry*; D005453:Fluorescence; D044085:Microfluidics / Q000295:instrumentation / Q000379:methods; D018613:Microscopy, Confocal; D009690:Nucleic Acid Conformation
D001583:Benzoxazoles; D015342:DNA Probes; D004279:DNA, Viral; C120457:POPO 3