Design, Synthesis, and Amplification of DNA Pools for In Vitro Selection

Bradley Hall1, John M. Micheletti2, Pooja Satya2, Krystal Ogle2, Jack Pollard3, Andrew D. Ellington1

1 Department of Chemistry and Biochemistry, University of Texas, Austin, Texas, 2 Freshman Research Initiative, University of Texas, Austin, Texas, 3 3rd Millennium Corporation, Cambridge, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 24.2
DOI:  10.1002/0471142727.mb2402s88
Online Posting Date:  October, 2009
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Preparation of a random‐sequence DNA pool is presented. The degree of randomization and the length of the random sequence are discussed, as is synthesis of the pool using a DNA synthesizer or via commercial synthesis companies. Purification of a single‐stranded pool and conversion to a double‐stranded pool are presented as step‐by‐step protocols. Support protocols describe determination of the complexity and skewing of the pool, and optimization of amplification conditions. Curr. Protoc. Mol. Biol. 88:24.2.1‐24.2.27. © 2009 by John Wiley & Sons, Inc.

Keywords: In vitro selection; DNA pool synthesis; phosphoramidite DNA synthesis; randomization

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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Purification of a Random Sequence Pool
  • Support Protocol 1: Determining the Pool Complexity
  • Support Protocol 2: Determining the Pool Bias
  • Support Protocol 3: Small‐Scale PCR Optimization of Pool Amplification
  • Basic Protocol 2: Large‐Scale PCR Amplification of Pool DNA
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Purification of a Random Sequence Pool

  • DNA pool
  • Ammonium hydroxide
  • n‐butanol
  • TE buffer, pH 8.0 ( appendix 22)
  • 2× denaturing dye (see recipe)
  • 3 M sodium acetate ( appendix 22)
  • Ethanol
  • Lyophilizer
  • 75° and 90°C water baths
  • 50‐ml Sterile Conical Tube Filter Unit (Thermo Scientific Nalgene)
  • Fluorescent TLC plate (VWR), wrapped in plastic wrap
  • UV lamp
  • Razor blades
  • Small‐bore syringes
  • 13‐ml centrifuge tubes capable of withstanding temperature extremes (Sarstedt)
  • Rotary shaker
  • Additional reagents and equipment for denaturing polyacrylamide gel electrophoresis (e.g., unit 2.12)

Support Protocol 1: Determining the Pool Complexity

  • Purified ssDNA pool
  • PCR primers
  • T4 polynucleotide kinase and buffer (New England Biolabs)
  • [γ‐32P]ATP (>3000 Ci/mmol)
  • 0.5 M EDTA, pH 8.0 ( appendix 22)
  • 3 M sodium acetate ( appendix 22)
  • 25:24:1 phenol/chloroform/isoamyl alcohol saturated with 10 mM Tris⋅Cl, pH 8.1/1 mM EDTA (see unit 2.1 or purchase from Sigma)
  • 3.0 M sodium acetate
  • 70% and 95% ethanol
  • TE buffer, pH 8.0 ( appendix 22)
  • 1 mg/ml blue‐dyed glycogen (GlycoBlue; Ambion)
  • 10× PCR amplification buffer (see recipe)
  • Taq DNA polymerase
  • 2× denaturing dye (see recipe)
  • Thermal cycler
  • 15 cm × 17 cm × 0.75 mm denaturing polyacrylamide gel (unit 2.12)
  • Phosphor imager plate and phosphor imager ( appendix 3A)
  • Additional reagents and equipment for quantitation of DNA (e.g., appendix 3D), end‐labeling of DNA (e.g., unit 3.10), phenol/chloroform and chloroform extraction of DNA (unit 2.1), PCR amplification (e.g., Chapter 15), denaturing polyacrylamide gel electrophoresis (unit 2.12), and phosphor imaging ( appendix 3A)

Support Protocol 2: Determining the Pool Bias

  • Purified ssDNA pool
  • PCR primers
  • PCR amplification buffer (see recipe) containing 1.5 mM Mg2 +
  • dNTP mix (dATP, dCTP, dGTP, dTTP; unit 3.4)
  • Taq DNA polymerase (e.g., New England Biolabs)
  • 3.8% NuSieve 3:1 agarose gel (Cambrex; also see unit 2.5)
  • 1× TBE buffer ( appendix 22)
  • dsDNA mass markers (e.g., Invitrogen)
  • Thermal cycler
  • Densitometer
  • Additional reagents and equipment for PCR (Chapter 15) and agarose gel electrophoresis (e.g., unit 2.5)

Support Protocol 3: Small‐Scale PCR Optimization of Pool Amplification

  • Purified ssDNA pool and primers
  • 0.5 M EDTA, pH 8.0 ( appendix 22)
  • 2‐butanol (for larger volumes)
  • 3 M sodium acetate
  • Ethanol
  • TE buffer, pH 8.0 ( appendix 22), containing 50 mM of a salt such as KCl
  • Thermal cycler or three water baths (one must be a circulating water bath)
  • 96‐well PCR plate or 13‐ml thermostable tubes (Sarstedt)
  • Thermometer
  • Styrofoam racks
  • Spectrophotometer or fluorimeter
  • Additional reagents and equipment for PCR amplification (unit 15.1; see protocol 4 for determination of conditions on a small scale) and phenol/chloroform and chloroform extraction of DNA (unit 2.1)
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Literature Cited

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