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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

1Department of Chemistry and Biochemistry, University of Texas, Austin, Texas
2Freshman Research Initiative, University of Texas, Austin, Texas
33rd 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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Abstract

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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Materials

Basic Protocol 1: Purification of a Random Sequence Pool

 Materials
  • DNA pool
  • Ammonium hydroxide
  • n-butanol
  • TE buffer, pH 8.0 (appendix 2)
  • 2× denaturing dye (see recipe)
  • 3 M sodium acetate (appendix 2)
  • 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

 Materials
  • Purified ssDNA pool
  • PCR primers
  • T4 polynucleotide kinase and buffer (New England Biolabs)
  • [-32P]ATP (>3000 Ci/mmol)
  • 0.5 M EDTA, pH 8.0 (appendix 2)
  • 3 M sodium acetate (appendix 2)
  • 25:24:1 phenol/chloroform/isoamyl alcohol saturated with 10 mM Tris×Cl, pH 8.1/1 mM EDTA (see unit 2.1A or purchase from Sigma)
  • 3.0 M sodium acetate
  • 70% and 95% ethanol
  • TE buffer, pH 8.0 (appendix 2)
  • 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 2.1A), PCR amplification (e.g., Chapter 15), denaturing polyacrylamide gel electrophoresis (unit 2.12), and phosphor imaging (appendix 3A)

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

 Materials
  • 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 2)
  • 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)

Basic Protocol 2: Large-Scale PCR Amplification of Pool DNA

 Materials
  • Purified ssDNA pool and primers
  • 0.5 M EDTA, pH 8.0 (appendix 2)
  • 2-butanol (for larger volumes)
  • 3 M sodium acetate
  • Ethanol
  • TE buffer, pH 8.0 (appendix 2), 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 Support Protocol 3 for determination of conditions on a small scale) and phenol/chloroform and chloroform extraction of DNA (unit 2.1A)
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Figures

  • Figure 24.2.1
    Flow chart outlining pool design, synthesis, and large-scale amplification.

    View Image
  • Figure 24.2.2
    Two examples of pools used in in vitro selection. Primers are shown above and below the sequence of the pool. The T7 promoter is delineated in bold. Restriction sites are underlined, with their enzymes listed.

    View Image
  • Figure 24.2.3
    Comparison of substitution distributions for a 66-nucleotide pool doped to either 18% or 35%.

    View Image
  • Figure 24.2.4
    Typical extension reaction. The pool used (N59) is shown to the right, next to the figure of the gel. Lane 1 shows the fully extended product and a large number of extensions on incomplete or damaged templates. Lane 2 is a control reaction containing only the primer. The extension reaction was incubated for 30 min.

    View Image
  • Figure 24.2.5
    A PCR cycle course and optimization of annealing temperature. The gel follows amplification of the N73 pool across a gradient of annealing temperatures. Two different pool synthesis methods were analyzed. Samples were removed after 0, 2, 4, 6 and 8, cycles. The pool used in the cycle course is depicted below the figure of the gel. IDT: Integrated DNA Technologies (http://www.idtdna.com/).

    View Image

Literature Cited

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