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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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 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

  Materials
  • 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

  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 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

  Materials
  • 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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Figures

Videos

Literature Cited

Literature Cited
   Abd‐Elsalam, K.A. 2003. Bioinformatic tools and guideline for PCR primer design. Afr. J. Biotech. 2:91‐95.
   Bartel, D.P. and Szostak, J.W. 1993. Isolation of new ribozymes from a large pool of random sequences. Science 261:1411‐1418.
   Bartel, D.P., Zapp, M.L., Green, M.R., and Szostak, J.W. 1991. HIV‐1 Rev regulation involves recognition of non‐Watson‐Crick base pairs in viral RNA. Cell 67:529‐536.
   Baskerville, S., Zapp, M., and Ellington, A.D. 1995. High‐resolution mapping of the human T‐cell leukemia virus type 1 rex‐binding element by in vitro selection. J. Virol. 69:7559‐7569.
   Beaucage, S.L. and Caruthers, M.H. 1981. Deoxynucleoside phosphoramidites. A new class of key intermediates for deoxypolynucleotide synthesis. Tetrahedron Lett. 22:1859‐1862.
   Beaucage, S.L. and Caruthers, M. 2000. Synthetic strategies and parameters involved in the synthesis of oligodeoxyribonucleotides according to the phosphoramidite method. Curr. Protoc. Nucl. Acid Chem. 00:3.3.1‐3.3.20.
   Beaucage, S.L. and Iyer, R.P. 1992. Advances in the synthesis of oligonucleotides by the phosphoramidite approach. Tetrahedron 48:2223‐2311.
   Boutros, R., Stokes, N., Bekaert, M., and Teeling, E.C. 2009. UniPrime2: A web service providing easier Universal Primer design. Nucl. Acids Res. 37:wx209‐w213.
   Breaker, R.R. 1997. In vitro selection of catalytic polynucleotides. Chem. Rev. 97:371‐390.
   Brown, D.M. 1993. A brief history of oligonucleotide synthesis. Methods Mol. Biol. 20:1‐17.
   Chandra, S. and Gopinath, B. 2007. Methods developed for SELEX. Anal. Bioanal. Chem. 387:171‐182.
   Chen, C.K. 2007. Complex SELEX against target mixture: Stochastic computer model, simulation, and analysis. Comput. Methods Programs Biomed. 87:189‐200.
   Chen, Z. and Ruffner, D.E. 1996. Modified crush‐and‐soak method for recovering oligodeoxynucleotides from polyacrylamide gel. BioTechniques 21:820‐822.
   Conrad, R., Keranen, L.M., Ellington, A.D., and Newton, A.C. 1994. Isozyme‐specific inhibition of protein kinase C by RNA aptamers. J. Biol. Chem. 269:32051‐32054.
   Crameri, A. and Stemmer, W.P.C. 1993. 1020‐fold aptamer library amplification without gel purification. Nucl. Acids Res. 21:4410.
   Fitzwater, T. and Polisky, B. 1996. A SELEX primer. Methods Enzymol. 267:275‐301.
   Giver, L., Bartel, D., Zapp, M., Pawul, A., Green, M., and Ellington, A.D. 1993. Selective optimization of the Rev‐binding element of HIV‐1. Nucl. Acids Res. 21:5509‐5516.
   Gold, L., Polisky, B., Uhlenbeck, O., and Yarus, M. 1995. Diversity of oligonucleotide functions. Annu. Rev. Biochem. 64:763‐797.
   Hermes, J.D., Parekh, S.M., Blacklow, S.C., Koster, H., and Knowles, J.R. 1989. A reliable method for random mutagenesis: The generation of mutant libraries using spiked oligodeoxyribonucleotide primers. Gene 84:143‐151.
   Hesselberth, J.R., Miller, D., Robertus, J., and Ellington, A.D. 2000. In vitro selection of RNA molecules that inhibit the activity of ricin a‐chain. J. Biol. Chem. 275:4937‐4942.
   Iyer, R.P. and Beaucage, S.L. 1999. Oligonucleotide synthesis. In Comprehensive Natural Products Chemistry, Vol. 7: DNA and Aspects of Molecular Biology (E.T. Kool, ed.) pp. 105‐152. Elsevier, London.
   Jaeger, J.A., Turner, D.H., and Zuker, M. 1989. Predicting optimal and suboptimal secondary structure for RNA. Methods Enzymol. 183:281‐306.
   Jaeger, L. 1997. The new world of ribozymes. Curr. Opin. Struct. Biol. 7:324‐335.
   Kim, N., Gan, H.H., and Schlick, T. 2007. A computational proposal for designing structured RNA pools for in vitro selection of RNAs. RNA. 13:478‐492.
   Legiewicz, M., Lozupone, C., Knight, R., and Yarus, M. 2005. Size, constant sequences, and optimal selection. RNA 11:1701‐1709.
   Lorsch, J.R. and Szostak, J.W. 1994. In vitro evolution of new ribozymes with polynucleotide kinase activity. Nature 371:31‐36.
   Lyamichev, V., Brow, M.A., and Dahlberg, J.E. 1993. Structure‐specific endonucleolytic cleavage of nucleic acids by eubacterial DNA polymerases. Science 260:778‐783.
   Michelson, A.M. and Todd, A.R. 1955. Nucleotides. XXXII. Synthesis of a dithymidine dinucleotide containing a 3′,5′‐internucleotidic linkage. J. Chem. Soc. 2632‐2638.
   Milligan, J.F., Groebe, D.R., Witherell, G.W., and Uhlenbeck, O.C. 1987. Oligoribonucleotide synthesis using T7 RNA polymerase and synthetic DNA templates. Nucl. Acids Res. 15:8783‐8798.
   Oliphant, A.R. and Struhl, S. 1989. An efficient method for generating proteins with altered enzymatic properties: application to beta‐lactamase. Proc. Natl. Acad. Sci. 86:9094‐9098.
   Oliphant, A.R., Brandl, C.J., and Struhl, K. 1990. Defining the sequence specificity of DNA‐binding proteins by selecting binding sites from random‐sequence oligonucleotides: Analysis of yeast GCN4 protein. Mol. Cell Biol. 9:2944‐2949.
   Pan, W. and Clawson, G.A. 2008. Catalytic DNAzymes: Derivations and functions. Expert Opin. Biol. Ther. 8:1071‐1085.
   Pan, W. and Clawson, G.A. 2009. The shorter the better: Reducing fixed primer regions of oligonucleotide libraries for aptamer selection. Molecules. 14:1353‐1369.
   PerSeptive Biosystems. 1998. Expedite Nucleic Acid Synthesis System: User's Guide. PerSeptive Biosystems, Framingham, Mass.
   Piasecki, S.K., Hall, B., and Ellington, A.D. 2009. Nucleic acid pool preparation and characterization. Methods Mol. Biol. 535:3‐18.
   Piganeau, N. 2009. In vitro selection of allosteric ribozymes. Methods Mol. Biol. 535:45‐57.
   Reese, C.B. 2005. Oligo‐ and poly‐nucleotides: 50 years of chemical synthesis. Org. Biomol. Chem. 3:3851‐3868.
   Sabeti, P.C., Unrau, P.J., and Bartel, D.P. 1997. Accessing rare activities from random RNA sequences: The importance of the length of molecules in the starting pool. Chem. Biol. 4:767‐774.
   Scott, W.G. 2007. Ribozymes. Curr. Opin. Struct. Biol. 17:280‐286.
   Singer, B.S., Shtatland, T., Brown, D., and Gold, L. 1997. Libraries for genomic SELEX. Nucl. Acids Res. 25:781‐786.
   Singh, V.K. and Kumar, A. 2001. PCR Primer Design. Mol. Biol. Today 2:27‐32.
   Stoltenburg, R., Reinemann, C., and Strehlitz, B. 2007. SELEX‐A (r)evolutionary method to generate high‐affinity nucleic acid ligands. Biomol. Eng. 24:381‐403.
   Strömberg, R. and Stawinski, J. 2004. Synthetic strategies and parameters involved in the synthesis of oligodeoxyribo‐ and oligoribonucleotides according to the H‐phosphonate method. Curr. Protoc. Nucl. Acid Chem. 19:3.4.1‐3.4.15.
   Tuerk, C. and Gold, L. 1990. Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science 249:505‐510
   Tuerk, C. and MacDougal‐Waugh, S. 1993. In vitro evolution of functional nucleic acids: High affinity RNA ligands of HIV‐1 proteins. Gene 137:33‐39.
   Unrau, P.J. and Bartel, D.P., 1998. RNA‐catalysed nucleotide synthesis. Nature. 395:260‐263.
   Vieux, E.F., Kwok, P.Y., and Miller, R.D. 2002. Primer design for PCR and sequencing in high‐throughput analysis of SNPs. Biotechniques 32:S28‐S32.
   Zon, G., Gallo, K.A., Samson, C.J., Shao, K., Summers, M.F., and Byrd, R.A. 1985. Analytical studies of “mixed sequence” oligodeoxyribonucleotides synthesized by competitive coupling of either methyl‐ or β‐cyanoethyl‐N,N‐diisopropylamino phosphoramidite reagents, including 2′‐deoxyinosine. Nucl. Acids Res. 13:8181‐8196.
   Zuker, M. 2003. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res. 31:3406‐3415.
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