Direct and precise length measurement of single, stretched DNA fragments by dynamic molecular combing and STED nanoscopy
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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.
KeywordsDNA Dynamic molecular combing Length measurement STED nanoscopy
We thank Professor Stefan W. Hell of Max Planck Institute for Biophysical Chemistry for the permission to use the Imspector imaging program. This research was supported by the Global Frontier Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (NRF-2014M3A6A4075063). We also acknowledge the BK21 Plus Program and SNU Brain Fusion Grant.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
- 1.Crick FH. On protein synthesis. Symp Soc Exp Biol. 1958;12:138–63.Google Scholar
- 7.Raghunathan S, Ricard CS, Lohman TM, Waksman G. Crystal structure of the homo-tetrameric DNA binding domain of Escherichia coli single-stranded DNA-binding protein determined by multiwavelength x-ray diffraction on the selenomethionyl protein at 2.9-angstrom resolution. Proc Natl Acad Sci U S A. 1997;94(13):6652–7.CrossRefGoogle Scholar
- 14.Hanne J, Falk HJ, Gorlitz F, Hoyer P, Engelhardt J, Sahl SJ, et al. STED nanoscopy with fluorescent quantum dots. Nat Commun. 2015;6. doi: 10.1038/ncomms8127.
- 18.Sambrook J, Russell DW. Molecular cloning: a laboratory manual. CSHL Press; 2001.Google Scholar
- 19.Stanojcic S, Sollelis L, Kuk N, Crobu L, Balard Y, Schwob E, et al. Single-molecule analysis of DNA replication reveals novel features in the divergent eukaryotes Leishmania and Trypanosoma brucei versus mammalian cells. Sci Rep-Uk. 2016;6. doi: 10.1038/srep23142.
- 30.Figeys D, Arriaga E, Renborg A, Dovichi NJ. Use of the fluorescent intercalating dyes POPO-3, YOYO-3 and YOYO-1 for ultrasensitive detection of double-stranded DNA separated by capillary electrophoresis with hydroxypropylmethyl cellulose and non-cross-linked polyacrylamide. J Chromatogr A. 1994;669(1–2):205–16.CrossRefGoogle Scholar