Random‐Primed, Phi29 DNA Polymerase‐Based Whole Genome Amplification

John R. Nelson1

1 GE Global Research, Niskayuna, New York
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 15.13
DOI:  10.1002/0471142727.mb1513s105
Online Posting Date:  January, 2014
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Abstract

Whole‐genome amplification by multiple displacement amplification (MDA) is a patented method to generate potentially unlimited genomic material when researchers are challenged with trace samples, or the amount of genomic DNA required for analysis exceeds the amount on hand. It is an isothermal reaction, using Phi29 DNA polymerase and random hexamer primers for unbiased amplification of linear DNA molecules, such as genomic DNA. The random‐primed MDA reaction provides extensive amplification coverage of the genome, generates extremely long DNA products, and provides high DNA yields. This unit explains the reaction, and describes use of the commercial kits available. Curr. Protoc. Mol. Biol. 105:15.13.1‐15.13.16. © 2014 by John Wiley & Sons, Inc.

Keywords: multiple displacement amplification (MDA); whole‐genome amplification (WGA); Phi29 DNA polymerase; isothermal amplification

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Random‐Primed, Whole‐Genome Amplification of Genomic DNA: The GenomiPhi Amplification Kit
  • Alternate Protocol 1: Whole‐Genome Amplification Directly from Blood or Cells: The GenomiPhi Amplification Kit
  • Basic Protocol 2: Random‐Primed, Whole‐Genome Amplification of Genomic DNA: The REPLI‐g Mini Kit
  • Alternate Protocol 2: Random‐Primed, Whole‐Genome Amplification of Genomic DNA from Blood or Cells: The REPLI‐g Mini Kit
  • Support Protocol 1: Quantification of Amplification Products
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Random‐Primed, Whole‐Genome Amplification of Genomic DNA: The GenomiPhi Amplification Kit

  Materials
  • Sample genomic DNA
  • illustra GenomiPhi V2 DNA amplification kit (http://www.gehealthcare.com/lifesciences) containing:
    • Sample buffer
    • Reaction buffer
    • Enzyme mix
    • Control DNA (lambda DNA)
  • TE (see recipe)
  • 0.5‐ml plastic microcentrifuge tubes
  • 30°, 65°, and 95°C water baths or thermal cycler
  • 30°C incubator

Alternate Protocol 1: Whole‐Genome Amplification Directly from Blood or Cells: The GenomiPhi Amplification Kit

  Materials
  • Cell sample in PBS (see recipe for PBS)
  • Denaturation solution (see recipe)
  • Neutralization buffer (see recipe)
  • GenomiPhi V2 DNA amplification kit (http://www.gehealthcare.com/lifesciences) containing:
    • Sample buffer
    • Reaction buffer
    • Enzyme mix
    • Control DNA (lambda DNA)
  • 0.5‐ml microcentrifuge tubes
  • 30°, 65°, and 95°C thermal cycler or water baths

Basic Protocol 2: Random‐Primed, Whole‐Genome Amplification of Genomic DNA: The REPLI‐g Mini Kit

  Materials
  • REPLI‐g Mini kits (http://www.qiagen.com):
    • Buffer DLB (reconstitute with 500 µl nuclease‐free water, mix well, and centrifuge briefly)
    • Stop solution
    • REPLI‐g mini reaction buffer
    • REPLI‐g mini DNA polymerase
  • Nuclease‐free water
  • Template DNA
  • 0.5‐ml microcentrifuge tubes
  • Vortex
  • Centrifuge
  • 30°, 65°, and 95°C thermal cycler or water baths

Alternate Protocol 2: Random‐Primed, Whole‐Genome Amplification of Genomic DNA from Blood or Cells: The REPLI‐g Mini Kit

  Materials
  • REPLI‐g Mini kits (http://www.qiagen.com) containing:
    • Buffer DLB (reconstitute in 500 µl nuclease‐free water, mix well, and centrifuge briefly)
    • Stop solution
    • REPLI‐g Mini reaction buffer
    • REPLI‐g Mini DNA polymerase
  • 1 M DTT (see recipe)
  • Template DNA (cellular material in PBS)
  • Nuclease‐free water
  • 0.5‐ml microcentrifuge tubes
  • 30°, 65°, and 95°C thermal cycler or water baths

Support Protocol 1: Quantification of Amplification Products

  Materials
  • Quant‐iT PicoGreen dsDNA quantification kit (Invitrogen, cat. no. P7581) containing:
    • 20× TE
    • Quant‐iT PicoGreen dsDNA quantification reagent
    • Lambda DNA standard
  • Sterile, distilled, DNase‐free water
  • Amplification products
  • Microplate
  • Foil
  • Centrifuge with a microplate adapter rotor
  • Fluorescence microplate reader
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Figures

Videos

Literature Cited

  Blanco, L. and Salas, M. 1984. Characterization and purification of a phage Phi29‐encoded DNA polymerase required for the initiation of replication. Proc. Natl. Acad. Sci. U.S.A. 81:5325‐5329.
  Blanco, L. and Salas, M. 1985. Characterization of a 3′‐5′ exonuclease activity in the phage Phi29‐encoded DNA polymerase. Nucleic Acids Res. 13:1239‐1249.
  Blanco, L., Bernad, A., Lazaro, J.M., Martin, G., Garmendia, C., and Salas, M. 1989. Highly efficient DNA synthesis by the phage Phi29 DNA polymerase. Symmetrical mode of DNA replication. J. Biol. Chem. 264:8935‐8940.
  Canceill, D., Viguera, E., and Ehrlich, S.D. 1999. Replication slippage of different DNA polymerases is inversely related to their strand displacement efficiency. J. Biol. Chem. 274:27481‐27490.
  de Vega, M., Lazaro, J.M., Salas, M., and Blanco, L. 1996. Primer terminus stabilization at the 3′‐5′ exonuclease active site of Phi29 DNA polymerase. Involvement of two amino acid residues highly conserved in proofreading DNA polymerases. EMBO J. 15:1182‐1192.
  de Vega, M., Ilyina, T., Lazaro, J.M., Salas, M., and Blanco, L. 1997. An invariant lysine residue is involved in catalysis at the 3′‐5′ exonuclease active site of eukaryotic‐type DNA polymerases. J. Mol. Biol. 270:65‐78.
  Dean, F.B., Nelson, J.R., Giesler, T.L., and Lasken, R.S. 2001. Rapid amplification of plasmid and phage DNA using Phi29 DNA polymerase and multiply‐primed rolling circle amplification. Genome Res. 11:1095‐1099.
  Dean, F.B., Hosono, S., Fang, L., Wu, X., Faruqi, A.F., Bray‐Ward, P., Sun, Z., Zong, Q., Du, Y., Du, J., Driscoll, M., Song, W., Kingsmore, S.F., Egholm, M., and Lasken, R.S. 2002. Comprehensive human genome amplification using multiple displacement amplification. Proc. Natl. Acad. Sci. U.S.A. 99:5261‐5266.
  Esteban, J.A., Soengas, M.S., Salas, M., and Blanco, L. 1994. 3′‐5′ Exonuclease active site of Phi29 DNA polymerase. Evidence favoring a metal ion‐assisted reaction mechanism. J. Biol. Chem. 269:31946‐31954.
  Garmendia, C., Bernad, A., Esteban, J.A., Blanco, L., and Salas, M. 1992. The bacteriophage Phi29 DNA polymerase, a proofreading enzyme. J. Biol. Chem. 267:2594‐2599.
  Hutchison, C.A. 3rd, Smith H.O., Pfannkoch, C., and Venter, J.C. 2005. Cell‐free cloning using Phi29 DNA polymerase. Proc. Natl. Acad. Sci. U.S.A. 102:17332‐17336
  Kool, E.T. 1996. Circular oligonucleotides: New concepts in oligonucleotide design. Annu. Rev. Biophys. Biomol. Struct. 25:1‐28.
  Kornberg, A. and Baker, T.A. 1992. DNA Replication. pp. 142‐153 and pp. 502‐503. W.H. Freeman and Co., New York.
  Kumar, G., Garnova, E., Reagin, M., and Vidali, A. 2008. Improved multiple displacement amplification with Phi29 DNA polymerase for genotyping of single human cells. Biotechniques 44:879‐890.
  Lasken, R.S. and Stockwell, T.B. 2007. Mechanism of chimera formation during the Multiple Displacement Amplification reaction. BMC Biotechnol. 7:19.
  Lasken, R.S., Dean, F.B., and Nelson, J.R. 2001. Multiply‐primed amplification of nucleic acid sequences. United States Patent 6,323,009.
  Lechner, R.L., Engler, M.J., and Richardson, C.C. 1983. Characterization of strand displacement synthesis catalyzed by bacteriophage T7 DNA polymerase. J. Biol. Chem. 258:11174‐11184.
  Lovmar, L. and Syvänen, A.C. 2006. Multiple displacement amplification to create a long‐lasting source of DNA for genetic studies. Hum. Mutat. 27:603‐614.
  Marcy, Y., Ishoey, T., Lasken, R.S., Stockwell, T.B., Walenz, B.P., Halpern, A.L., Beeson, K.Y., Goldberg, S.M., and Quake, S.R. 2007. Nanoliter reactors improve multiple displacement amplification of genomes from single cells. PLoS Genet. 3:1702‐1708.
  Nelson, J.R., Cai, Y.C., Giesler, T.L., Farchaus, J.W., Sundaram, S.T., Ortiz‐Rivera, M., Hosta, L.P., Hewitt, P.L., Mamone, J.A., Palaniappan, C., and Fuller, C.W. 2002. TempliPhi, Phi29 DNA polymerase–based rolling circle amplification of templates for DNA sequencing. Biotechniques 32:S44‐S47.
  Raghunathan, A., Ferguson, H.R. Jr., Bornarth, C.J., Song, W., Driscoll, M., and Lasken, R.S. 2005. Genomic DNA amplification from a single bacterium. Appl. Environ. Microbiol. 71:3342‐3347.
  Spits, C., Le Caignec, C., De Rycke, M., Van Haute, L., Van Steirteghem, A., Liebaers, I., and Sermon, K. 2006. Optimization and evaluation of single‐cell whole‐genome multiple displacement amplification. Hum. Mutat. 27:496‐503.
  Zhang, K., Martiny, A.C., Reppas, N.B., Barry, K.W., Malek, J., Chisholm, S.W., and Church, G.M. 2006. Sequencing genomes from single cells by polymerase cloning. Nat. Biotechnol. 6:680‐686.
Internet Resources
  http://www.gelifesciences.com
  For more information on the GenomiPhi product line, new kits and specialty applications, and customized workflows.
  http://www.qiagen.com
  For more information on the Repli‐g product line, new kits and specialty applications, and customized workflows.
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