Isolation of Single‐Stranded DNA

Yuji Wakimoto1, Jianming Jiang1, Hiroko Wakimoto1

1 Department of Genetics, Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 2.15
DOI:  10.1002/0471142727.mb0215s107
Online Posting Date:  July, 2014
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Abstract

Single‐stranded DNA (ssDNA) is often used for DNA sequencing as well as microarray and hybridization technologies. Asymmetric PCR or exonuclease digestion followed by urea gel separation and isolation on streptavidin‐coated magnetic beads are commonly used for this purpose. These two methods may not yield large amounts of highly purified ssDNA. This protocol for ssDNA isolation from PCR‐amplified DNA involves biotin labeling of one strand and subsequent strand separation utilizing streptavidin‐coated magnetic beads. Curr. Protoc. Mol. Biol. 107:2.15.1‐2.15.9. © 2014 by John Wiley & Sons, Inc.

Keywords: single‐stranded DNA; biotin; streptavidin; magnetic beads

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: PCR Amplification and Amplicon Purification
  • Alternate Protocol 1: Pippin Prep Purification of PCR Products
  • Basic Protocol 2: Isolation of ssDNA from dsDNA with Streptavidin‐Coated Magnetic Beads
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: PCR Amplification and Amplicon Purification

  Materials
  • DNA template
  • 10 µM forward primer corresponding to desired strand
  • 10 µM reverse strand biotinylated‐primer corresponding to undesired strand that will be removed after the isolation
  • 10 mM 4dNTP mix (prepared from 100 mM stock)
  • 2 U/µl Phusion high‐fidelity (HF) DNA polymerase with 5× buffer (NEB, cat. no. M0530S, stored at −20°C)
  • Nuclease‐free H 2O (Ambion, cat. no. AM9937)
  • QIAquick PCR purification kit (Qiagen, cat. no. 28104, stored at room temperature), including spin columns, 2‐ml collection tubes, Buffers PB, PE, and EB, and 3 M sodium acetate
  • 96% to 100% ethanol (required for Qiagen kit, add to Buffer PE as per QIAquick instructions)
  • TE buffer (Ambion, cat. no. AM9849 or , optional)
  • Thermal cycler
NOTE: If the PCR sample is expected to contain nonspecific products, it is strongly recommended to perform electrophoresis followed by gel extraction (unit ) or to use Pippin Prep as described below in Alternate Protocol 1.

Alternate Protocol 1: Pippin Prep Purification of PCR Products

  Additional Materials (also see protocol 1)
  • Pippin Gel Cassettes (3% agarose, with ethidium bromide; Sage Science, cat. no. CSD3010), kit includes Pippin electrophoresis buffer, Pippin Marker B, Pippin running buffer, and adhesive tape strip
  • Pippin Prep DNA size selection system (Sage Science)

Basic Protocol 2: Isolation of ssDNA from dsDNA with Streptavidin‐Coated Magnetic Beads

  Materials
  • Melt solution, freshly prepared (see recipe)
  • Neutralization solution, freshly prepared (see recipe)
  • Dynabeads® M‐270 streptavidin (Invitrogen, cat. no. 65305, stored at 4°C)
  • SureSelect binding buffer (Agilent, cat. no. G9607A, stored at room temperature)
  • Purified PCR product (from protocol 1 or protocol 2Alternate Protocol)
  • 20 mg/ml glycogen (Roche Applied Science, cat. no. 10901393001, stored at −20°C)
  • Isopropanol (stored at room temperature)
  • Ice‐cold 70% ethanol
  • Nuclease‐free water (Ambion, cat. no. AM9937)
  • Nonstick RNase‐free microcentrifuge tubes, 1.5 ml (Ambion, cat. no. AM12450)
  • Magnetic bead separator (DynaMag™‐2 Magnet, Invitrogen, cat. no. 12321D)
NOTE: Prepare melt solution and neutralization solution fresh before proceeding.
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Figures

Videos

Literature Cited

Literature Cited
   Ageno, M. , Dore, E. , and Frontali, C. 1969. The alkaline denaturation of DNA. Biophys. J. 9:1281‐1311.
   Avci‐Adali, M. , Paul, A. , Wilhelm, N. , Ziemer, G. , and Wendel, H.P. 2009. Upgrading SELEX technology by using lambda exonuclease digestion for single‐stranded DNA generation. Molecules 15:1‐11.
   Barbas, C.F. III , Burton, D.R. , Scott, J.R. , and Silverman, G.J. 2007. Quantitation of DNA and RNA. Cold Spring Harb. Protoc. doi:10.1101/pdb.ip47.
   Beaulieu, M. , Larson, G.P. , Geller, L. , Flanagan, S.D. , and Krontiris, T.G. 2001. PCR candidate region mismatch scanning: Adaptation to quantitative, high‐throughput genotyping. Nucleic Acids Res. 29:1114‐1124.
   Beliveau, B.J. , Joyce, E.F. , Apostolopoulos, N. , Yilmaz, F. , Fonseka, C.Y. , McCole, R.B. , Chang, Y. , Li, J.B. , Senaratne, T.N. , Williams, B.R. , Rouillard, J.M. , and Wu, C.T. 2013. Versatile design and synthesis platform for visualizing genomes with Oligopaint FISH probes. Proc. Natl. Acad. Sci. U.S.A. 109:21301‐21306.
   Citartan, M. , Tang, T.H. , Tan, S.C. , and Gopinath, S.C. 2011. Conditions optimized for the preparation of single‐stranded DNA (ssDNA) employing lambda exonuclease digestion in generating DNA aptamer. World J. Microbiol. Biotechnol. 27:1167‐1173.
   Green, N.M. 1975. Avidin. Adv. Protein Chem. 29:85‐133.
   Hansen‐Hagge, T.E. , Trefzer, U. , zu Reventlow, A.S. , Kaltoft, K. , and Sterry, W. 2001. Identification of sample‐specific sequences in mammalian cDNA and genomic DNA by the novel ligation‐mediated subtraction (Limes). Nucleic Acids Res. 29:E20.
   Hultman, T. , Ståhl, S. , Hornes, E. , and Uhlén, M. 1989. Direct solid phase sequencing of genomic and plasmid DNA using magnetic beads as solid support. Nucleic Acids Res. 17:4937‐4946
   Hultman, T. , Bergh, S. , Moks, T. , and Uhlén, M. 1991. Bidirectional solid‐phase sequencing of in vitro‐amplified plasmid DNA. Biotechniques 10:84‐93.
   Laveder, P. , De Pittà, C. , Toppo, S. , Valle, G. , and Lanfranchi, G. 2002. A two‐step strategy for constructing specifically self‐subtracted cDNA libraries. Nucleic Acids Res. 30:E38.
   Maricic, T. and Pääbo, S. 2009. Optimization of 454 sequencing library preparation from small amounts of DNA permits sequence determination of both DNA strands. Biotechniques 46:51‐52, 54‐57.
   Paul, A , Avci‐Adali, M , Ziemer, G , and Wendel, HP . 2009. Streptavidin‐coated magnetic beads for DNA strand separation implicate a multitude of problems during cell‐SELEX. Oligonucleotides 19:243‐254.
   Pourmand, N. , Elahi, E. , Davis, R.W. , and Ronaghi, M. 2002. Multiplex pyrosequencing. Nucleic Acids Res. 30:e31.
   Pradel, N. , Leroy‐Setrin, S. , Joly, B. , and Livrelli, V. 2002. Genomic subtraction to identify and characterize sequences of Shiga toxin‐producing Escherichia coli O91:H21. Appl. Environ. Microbiol. 68:2316‐2325.
   Svobodová, M. , Pinto, A. , Nadal, P. , and O'Sullivan, C.K. 2012. Comparison of different methods for generation of single‐stranded DNA for SELEX processes. Anal. Bioanal. Chem. 404:835‐842.
   Trower, M.K. 1996. Preparation of ssDNA from phagemid vectors. Methods Mol. Biol. 58:469‐476.
   von Wintzingerode, F ., Böcker, S. , Schlötelburg, C. , Chiu, N.H. , Storm, N. , Jurinke, C. , Cantor, C.R. , Göbel, U.B. , and van den Boom, D. 2002. Base‐specific fragmentation of amplified 16S rRNA genes analyzed by mass spectrometry: a tool for rapid bacterial identification. Proc. Natl. Acad. Sci. U.S.A. 99:7039‐7044.
   Wilson, R. 2011. Preparation of single‐stranded DNA from PCR products with streptavidin magnetic beads. Nucleic Acid Ther. 21:437‐440.
   Wooddell, C.I. and Burgess, R.R. 1996. Use of asymmetric PCR to generate long primers and single‐stranded DNA for incorporating cross‐linking analogs into specific sites in a DNA probe. Genome Res. 6:886‐892.
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