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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 Nucleic Acid Chemistry
Unit Number: 
Unit 9.2
DOI: 
10.1002/0471142700.nc0902s39
Online Posting Date: 
December, 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. Nucleic Acid Chem. 39:9.2.1-9.2.28. © 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 2A)
  • 2× denaturing dye (see recipe)
  • 3 M sodium acetate (appendix 2A)
  • 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., appendix 3B or unit 10.4)

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 2A)
  • 3 M sodium acetate (appendix 2A)
  • 25:24:1 phenol/chloroform/isoamyl alcohol saturated with 10 mM Tris×Cl, pH 8.0/1 mM EDTA (appendix 2A or purchase from Sigma)
  • 70% and 95% ethanol
  • TE buffer, pH 8.0 (appendix 2A)
  • 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 (appendix 3B)
  • Phosphor imager plate and phosphor imager (appendix 3B)
  • Additional reagents and equipment for performing DNA quantitation (e.g., unit 5.2), DNA dephosphorylation (e.g., Tabor, 1987), phenol/chloroform and chloroform extraction of DNA (e.g., Moore and Dowhan, 2002), PCR amplification of DNA (e.g., Chapter 15, Ausubel et al., 2009), and denaturing polyacrylamide gel electrophoresis and phosphor imaging (appendix 3B)

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; appendix 2A)
  • Taq DNA polymerase (e.g., New England Biolabs)
  • 3.8% NuSieve 3:1 agarose gel (Cambrex; also see Voytas, 2000)
  • 1× TBE buffer (appendix 2A)
  • dsDNA mass markers (e.g., Invitrogen)
  • Thermal cycler
  • Densitometer
  • Additional reagents and equipment for PCR (Chapter 15, Ausubel et al., 2009) and agarose gel electrophoresis (e.g., Voytas, 2000)

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

 Materials
  • Purified ssDNA pool and primers
  • 0.5 M EDTA, pH 8.0 (appendix 2A)
  • 2-butanol (for larger volumes)
  • 3 M sodium acetate
  • Ethanol
  • TE buffer, pH 8.0 (appendix 2A), 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 performing PCR amplification (Kramer and Coen, 2001; see Support Protocol 3 for determination of conditions on a small scale) and phenol/chloroform and chloroform extraction of DNA (e.g., see Moore and Dowhan, 2002).
Looking for materials?  Suppliers Guide
     
 
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Figures

  • Figure 9.2.1
    Flow chart outlining pool design, synthesis, and large-scale amplification.

    View Image
  • Figure 9.2.2
    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 9.2.3
    Comparison of substitution distributions for a 66-nucleotide pool doped to either 18% or 35%.

    View Image
  • Figure 9.2.4
    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 9.2.5
    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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