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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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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Basic Protocol 1: PCR Amplification and Amplicon Purification

  • 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

  • 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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Literature Cited

Literature Cited
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