High-yield purification of exceptional-quality, single-molecule DNA substrates
Keywords:column chromatography, DNA substrate, oligonucleotide, single-molecule, TSKgel DNA-stat
Single-molecule studies involving DNA or RNA, require homogeneous preparations of nucleic acid substrates of exceptional quality. Over the past several years, a variety of methods have been published describing different purification methods but these are frustratingly inconsistent with variable yields even in the hands of experienced bench scientists. To address these issues, we present an optimized and straightforward, column-based approach that is reproducible and produces high yields of substrates or substrate components of exceptional quality. Central to the success of the method presented is the use of a non-porous anion exchange resin. In addition to the use of this resin, we encourage the optimization of each step in the construction of substrates. The fully optimized method produces high yields of a hairpin DNA substrate of exceptional quality. While this substrate is suitable for single-molecule, magnetic tweezer experiments, the described method is readily adaptable to the production of DNA substrates for the majority of single-molecule studies involving nucleic acids ranging in size from 70
Sun Z, Tan HY, Bianco PR, Lyubchenko YL. Remodeling of RecG Helicase at the DNA Replication Fork by SSB Protein. Sci Rep. 2015;5:9625. PMID: 25923319
Zhao X, Guo S, Lu C, Chen J, Le S, Fu H, et al. Single-molecule manipulation quantification of site-specific DNA binding. Curr Opin Chem Biol. 2019;53:106-17.
Kaur G, Lewis JS, van Oijen AM. Shining a Spotlight on DNA: Single-Molecule Methods to Visualise DNA. Molecules. 2019;24(3). PMID: 31677535
Neuman KC, Nagy A. Single-molecule force spectroscopy: optical tweezers, magnetic tweezers and atomic force microscopy. Nat Methods. 2008;5(6):491-505. PMID: 18511917
Hodeib S, Raj S, Manosas M, Zhang W, Bagchi D, Ducos B, et al. Single molecule studies of helicases with magnetic tweezers. Methods. 2016;105:3-15. PMID: 27371121
Kimura Y, Bianco PR. Single molecule studies of DNA binding proteins using optical tweezers. Analyst. 2006;131(8):868-74. PMID: 17028717
Bianco PR, Brewer LR, Corzett M, Balhorn R, Yeh Y, Kowalczykowski SC, et al. Processive translocation and DNA unwinding by individual RecBCD enzyme molecules. Nature. 2001;409(6818):374-8. PMID: 11201750
Maleki P, Budhathoki JB, Roy WA, Balci H. A practical guide to studying G-quadruplex structures using single-molecule FRET. Mol Genet Genomics. 2017;292(3):483-98. PMID: 28150040
Roy R, Hohng S, Ha T. A practical guide to single-molecule FRET. Nat Methods. 2008;5(6):507-16. PMID: 18511918
Manosas M, Perumal SK, Bianco PR, Ritort F, Benkovic SJ, Croquette V. RecG and UvsW catalyse robust DNA rewinding critical for stalled DNA replication fork rescue. Nat Commun. 2013;4:2368. PMID: 24013402
Lionnet T, Allemand JF, Revyakin A, Strick TR, Saleh OA, Bensimon D, et al. Single-molecule studies using magnetic traps. Cold Spring Harb Protoc. 2012;2012(1):34-49. PMID: 22194259
White KH, Visscher K. Optical trapping and unfolding of RNA. Methods Mol Biol. 2011;783:21-43. PMID: 21909881
Wen JD, Manosas M, Li PT, Smith SB, Bustamante C, Ritort F, et al. Force unfolding kinetics of RNA using optical tweezers. I. Effects of experimental variables on measured results. Biophys J. 2007;92(9):2996-3009. PMID: 17293410
Papini FS, Seifert M, Dulin D. High-yield fabrication of DNA and RNA constructs for single molecule force and torque spectroscopy experiments. Nucleic Acids Res. 2019;47(22):e144. PMID: 31584079
Goto Y, Akahori R, Yanagi I, Takeda KI. Solid-state nanopores towards single-molecule DNA sequencing. J Hum Genet. 2020;65(1):69-77. PMID: 31420594
Poh JJ, Gan SK. Comparison of customized spin-column and salt-precipitation finger-prick blood DNA extraction. Biosci Rep. 2014;34(5). PMID: 25222694
Pederson NE. Spin-column chromatography for DNA purification. Anal Biochem. 1996;239(1):117-8.
Nickoloff JA. Sepharose spin column chromatography. A fast, nontoxic replacement for phenol:chloroform extraction/ethanol precipitation. Mol Biotechnol. 1994;1(1):105-8. PMID: 7859149
Hourfar MK, Michelsen U, Schmidt M, Berger A, Seifried E, Roth WK. High-throughput purification of viral RNA based on novel aqueous chemistry for nucleic acid isolation. Clin Chem. 2005;51(7):1217-22. PMID: 15976102
Vandeventer PE, Mejia J, Nadim A, Johal MS, Niemz A. DNA adsorption to and elution from silica surfaces: influence of amino acid buffers. J Phys Chem B. 2013;117(37):10742-9. PMID: 23931415
Noirclerc-Savoye M, Gallet B, Bernaudat F, Vernet T. Large scale purification of linear plasmid DNA for efficient high throughput cloning. Biotechnol J. 2010;5(9):978-85. PMID: 20845387
Bernardi G. Chromatography of nucleic acids on hydroxyapatite. Nature. 1965;206(4986):779-83. PMID: 5840127
Shoyab M, Sen A. The isolation of extrachromosomal DNA by hydroxyapatite chromatography. Methods Enzymol. 1979;68:199-206. PMID: 232213
Lechner RL, Richardson CC. A preformed, topologically stable replication fork. Characterization of leading strand DNA synthesis catalyzed by T7 DNA polymerase and T7 gene 4 protein. J Biol Chem. 1983;258(18):11185-96. PMID: 6885816
Andrews-Pfannkoch C, Fadrosh DW, Thorpe J, Williamson SJ. Hydroxyapatite-mediated separation of double-stranded DNA, single-stranded DNA, and RNA genomes from natural viral assemblages. Appl Environ Microbiol. 2010;76(15):5039-45. PMID: 20543058
Westman E, Eriksson S, Laas T, Pernemalm PA, Skold SE. Separation of DNA restriction fragments by ion-exchange chromatography on FPLC columns Mono P and Mono Q. Anal Biochem. 1987;166(1):158-71. PMID: 2890317
Kim Y, de la Torre A, Leal AA, Finkelstein IJ. Efficient modification of lambda-DNA substrates for single-molecule studies. Sci Rep. 2017;7(1):2071. PMID: 28522818
Mueller SH, Spenkelink LM, van Oijen AM, Lewis JS. Design of customizable long linear DNA substrates with controlled end modifications for single-molecule studies. Anal Biochem. 2020;592:113541. PMID: 31870680
Luzzietti N, Brutzer H, Klaue D, Schwarz FW, Staroske W, Clausing S, et al. Efficient preparation of internally modified single-molecule constructs using nicking enzymes. Nucleic Acids Res. 2011;39(3):e15. PMID: 21071409
Moriyama H, Shimada M, Muranaka K, Iwaeda T. High Speed and high resolution anion exchange chromatography for biological samples on non-porous packings. HPLC; Baltimore, MD2008.
Kato Y, Yamasaki Y, Onaka A, Kitamura T, Hashimoto T, Murotsu T, et al. Separation of DNA restriction fragments by high-performance ion-exchange chromatography on a non-porous ion exchanger. J Chromatogr. 1989;478(1):264-8. PMID: 2532220
Tellez CM, Cole KD. Method for the characterization of size-exclusion chromatography mediafor preparative purification of DNA restriction fragments. Biotechnology Techniques. 1999;13:395-401. doi: 10.1023/A:1008932407733
Wysoczynski CL, Roemer SC, Dostal V, Barkley RM, Churchill ME, Malarkey CS. Reversed-phase ion-pair liquid chromatography method for purification of duplex DNA with single base pair resolution. Nucleic Acids Res. 2013;41(20):e194. PMID: 24013567
Abdulrahman A, Ghanem A. Recent advances in chromatographic purification of plasmid DNA for gene therapy and DNA vaccines: A review. Anal Chim Acta. 2018;1025:41-57. PMID: 29801607
Iuliano S, Fisher JR, Chen M, Kelly WJ. Rapid analysis of a plasmid by hydrophobic-interaction chromatography with a non-porous resin. J Chromatogr A. 2002;972(1):77-86. PMID: 12395948
Wang Y, Sun Z, Bianco PR, Lyubchenko YL. Atomic force microscopy-based characterization of the interaction of PriA helicase with stalled DNA replication forks. J Biol Chem. 2020;295(18):6043-52. PMID: 32209655
Liu X, Seet JX, Shi Y, Bianco PR. Rep and UvrD Antagonize One Another at Stalled Replication Forks and This Is Exacerbated by SSB. ACS Omega. 2019;4(3):5180-96. PMID: 30949615
How to Cite
Authors who publish with JBM agree to the following terms:
- Authors retain copyright and grant JBM right of first publication with the work simultaneously licensed under a Creative Commons Attribution License that allows others to share the work with an acknowledgement of the work's authorship and initial publication in this journal.
- Authors are able to enter into separate, additional contractual arrangements for the non-exclusive distribution of the journal's published version of the work (e.g., post it to an institutional repository or publish it in a book), with an acknowledgement of its initial publication in this journal.
- Authors are permitted and encouraged to post their work online (e.g., in institutional repositories or on their website) prior to and during the submission process, as it can lead to productive exchanges, as well as earlier and greater citation of published work (See The Effect of Open Access).