Direct and precise length measurement of single, stretched DNA fragments by dynamic molecular combing and STED nanoscopy

Abstract

A combination of DNA stretching method and super-resolution nanoscopy allows an accurate and precise measurement of the length of DNA fragments ranging widely in size from 117 to 23,130 bp. BstEII- and HindIII-treated λDNA fragments were stained with an intercalating dye and then linearly stretched on a coverslip by dynamic molecular combing. The image of individual DNA fragments was obtained by stimulated emission depletion nanoscopy. For DNA fragments longer than ∼1000 bp, the measured lengths of DNA fragments were consistently within ∼0.5 to 1.0 % of the reference values, raising the possibility of this method in a wide range of applications including facile detection for copy number variations and trinucleotide repeat disorder.

Quantifying Polymer Chain Orientation in Strong and Tough Nanofibers with Low Crystallinity: Toward Next Generation Nanostructured Superfibers

“…Similar approaches with better, subdiffraction limit resolution, can be undertaken using been used to examine spatial arrangement of protein nanofibrils, [235][236][237] measure the length of stretched DNA segments, 238 and for resolving the distances between adjacent fluorescent sites with spatial resolution unavailable in regular fluorescent microscopy. 239 It is likely that such approaches can be adopted to examine chain extension in electrospun NFs.…”

“…Similar approaches with better, subdiffraction limit resolution, can be undertaken using emerging super resolution microscopy techniques such as structured illumination microscopy, simulated emission depletion, stochastic optical reconstruction microscopy, or photoactivated localization microscopy. The techniques have already been used to examine spatial arrangement of protein nanofibrils, − measure the length of stretched DNA segments, and resolve the distances between adjacent fluorescent sites with spatial resolution unavailable in regular fluorescent microscopy . It is likely that such approaches can be adopted to examine chain extension in electrospun NFs.…”

Molecular Combing of Various Poly(n-Alkyl Acrylate) Chains on Mica by the Dipping Method

“…1−16 DNA can be molecularly combed by drying a DNA solution sandwiched between two glass plates 2−5 or by pulling up a substrate from a DNA solution (dipping method). 1,8,[11][12][13]15,16 DNA chains are believed to be combed by the capillary force of the receding meniscus that acts on the DNA chains during drying and dipping. The molecular combing of DNA has also been achieved by translating a droplet of DNA solution on a substrate 6,10,14 and by flowing a solution in microchannels.…”

“…Fixing a polymer chain on a substrate in a completely stretched state is called molecular combing . − If a polymer chain is molecularly combed, by direct observation with microscopy methods such as atomic force microscopy (AFM), − the chain could be analyzed more precisely than has been achieved with the measurement techniques available today. For example, the precise distribution of monomers in a copolymer, the precise distribution of the branch length, and the branch position in the main chain of a branched polymer could be characterized in detail.…”

“…For example, the precise distribution of monomers in a copolymer, the precise distribution of the branch length, and the branch position in the main chain of a branched polymer could be characterized in detail. The molecular combing of a rigid biopolymer, DNA, has been extensively studied for the purpose of mapping genetic information with fluorescence microscopy, AFM, and scanning tunneling microscopy. − DNA can be molecularly combed by drying a DNA solution sandwiched between two glass plates − or by pulling up a substrate from a DNA solution (dipping method). ,,− ,, DNA chains are believed to be combed by the capillary force of the receding meniscus that acts on the DNA chains during drying and dipping. The molecular combing of DNA has also been achieved by translating a droplet of DNA solution on a substrate ,, and by flowing a solution in microchannels. − In contrast, molecular combings of flexible polymers require a significant reduction in entropy during chain stretching, and therefore, we believe, no attempt for the molecular combing of flexible polymers has been reported for more than two decades since the first report of the DNA molecular combing.…”