Whole Genome Amplification with Phi29 DNA Polymerase to Enable Genetic or Genomic Analysis of Samples of Low DNA Yield

  • Kaisa Silander
  • Janna Saarela
Part of the Methods in Molecular Biology™ book series (MIMB, volume 439)


In many large genetic studies, the amount of available DNA can be one of the criteria for selecting samples for study. In the case of large population cohorts, selecting samples based on their DNA yield can lead to biased sample selection. In addition, many valuable clinical and research sample collections exist in which the amount of DNA is very small. Unbiased whole genome amplification (WGA) of such unique samples enables genomewide scale genetic studies that would have been impossible otherwise. Multiply primed rolling circle amplification (MPRCA) and multiple displacement amplification (MDA) methods are based on the same principle. The DNA amplification is non-PCR based and uses Φ29 DNA polymerase and random hexamer primers for unbiased whole genome amplification. MDA is used for linear DNA molecules, such as genomic DNA. This chapter reviews the various applications in which whole genome amplified DNA can be used, the types of commercial kits available, and the quality control steps necessary before using the DNA in the genetic studies.


multiply primed rolling circle amplification multiple displacement amplification WGA DNA yield genotyping 


  1. 1.
    1. Nelson DL, Ledbetter SA, Corbo L, Victoria MF, Ramirez-Solis R, Webster TD, Ledbetter DH, Caskey CT (1989) Alu polymerase chain reaction: A method for rapid isolation of human-specific sequences from complex DNA sources. Proc Natl Acad Sci USA 86:6686–6690CrossRefPubMedGoogle Scholar
  2. 2.
    2. Zhang L, Cui X, Schmitt K, Hubert R, Navidi W, Arnheim N (1992) Whole genome amplification from a single cell: Implications for genetic analysis. Proc Natl Acad Sci USA 89:5847–5851CrossRefPubMedGoogle Scholar
  3. 3.
    3. Telenius H, Carter NP, Bebb CE, Nordenskjold M, Ponder BA, Tunnacliffe A (1992) Degenerate oligonucleotide-primed PCR: General amplification of target DNA by a single degenerate primer. Genomics 13:718–725CrossRefPubMedGoogle Scholar
  4. 4.
    4. Dietmaier W, Hartmann A, Wallinger S, Heinmoller E, Kerner T, Endl E, Jauch KW, Hofstadter F, and Ruschoff J (1999) Multiple mutation analyses in single tumor cells with improved whole genome amplification. Am J Pathol 154:83–95CrossRefPubMedGoogle Scholar
  5. 5.
    5. Kittler R, Stoneking M, Kayser M (2002) A whole genome amplification method to generate long fragments from low quantities of genomic DNA. Anal Biochem 300:237–244CrossRefPubMedGoogle Scholar
  6. 6.
    6. Klein CA, Schmidt-Kittler O, Schardt JA, Pantel K, Speicher MR, Riethmuller G (1999) Comparative genomic hybridization, loss of heterozygosity, and DNA sequence analysis of single cells. Proc Natl Acad Sci USA 96:4494–4499CrossRefPubMedGoogle Scholar
  7. 7.
    7. Lucito R, Nakimura M, West JA, Han Y, Chin K, Jensen K, McCombie R, Gray JW, Wigler M (1998) Genetic analysis using genomic representations. Proc Natl Acad Sci USA 95:4487–4492CrossRefPubMedGoogle Scholar
  8. 8.
    8. Vos P, Hogers R, Bleeker M, Reijans M, van de Lee T, Homes M, Frijters A, Pot J, Peleman J, Kuiper M et al. (1995) AFLP: A new technique for DNA fingerprinting. Nucleic Acids Res 23:4407–1414CrossRefPubMedGoogle Scholar
  9. 9.
    9. Tanabe C, Aoyagi K, Sakiyama T, Kohno T, Yanagitani N, Akimoto S, Sakamoto M, Sakamoto H, Yokota J, Ohki M et al (2003) Evaluation of a whole-genome amplification method based on adaptor-ligation PCR of randomly sheared genomic DNA. Genes Chromosomes Cancer 38:168–176CrossRefPubMedGoogle Scholar
  10. 10.
    10. Langmore JP (2002) Rubicon Genomics, Inc. Pharmacogenomics 3:557–560CrossRefPubMedGoogle Scholar
  11. 11.
    11. Lovmar L, Syvanen AC (2006) Multiple displacement amplification to create a long-lasting source of DNA for genetic studies. Hum Mutat 27:603–614CrossRefPubMedGoogle Scholar
  12. 12.
    12. Dean FB, Hosono S, Fang L, Wu X, Faruqi AF, Bray-Ward P, Sun Z, Zong Q, Du Y, Du J et al (2002) Comprehensive human genome amplification using multiple displacement amplification. Proc Natl Acad Sci USA 99:5261–5266CrossRefPubMedGoogle Scholar
  13. 13.
    13. Hosono S, Faruqi AF, Dean FB, Du Y, Sun Z, Wu X, Du J, Kingsmore SF, Egholm M, Lasken RS (2003) Unbiased whole-genome amplification directly from clinical samples. Genome Res 13:954–964CrossRefPubMedGoogle Scholar
  14. 14.
    14. Lu Y, Gioia-Patricola L, Gomez JV, Plummer M, Franceschi S, Kato I, Canzian F (2005) Use of whole genome amplification to rescue DNA from plasma samples. Biotechniques 39:511–515CrossRefPubMedGoogle Scholar
  15. 15.
    15. Rook MS, Delach SM, Deyneko G, Worlock A, Wolfe JL (2004) Whole genome amplification of DNA from laser capture-microdissected tissue for high-throughput single nucleotide polymorphism and short tandem repeat genotyping. Am J Pathol 164:23–33CrossRefPubMedGoogle Scholar
  16. 16.
    16. Ballantyne KN, van Oorschot RA, Mitchell RJ (2007) Comparison of two whole genome amplification methods for STR genotyping of LCN and degraded DNA samples. Forensic Sci Int 166:35–41CrossRefPubMedGoogle Scholar
  17. 17.
    17. Short AD, Kennedy LJ, Forman O, Barnes A, Fretwell N, Wiggall R, Thomson W, and Oilier WE (2005) Canine DNA subjected to whole genome amplification is suitable for a wide range of molecular applications. J Hered 96:829–835CrossRefPubMedGoogle Scholar
  18. 18.
    18. Gorrochotegui-Escalante N, Black WCT (2003) Amplifying whole insect genomes with multiple displacement amplification. Insect Mol Biol 12:195–200CrossRefPubMedGoogle Scholar
  19. 19.
    19. Gadkar V, Rillig MC (2005) Suitability of genomic DNA synthesized by strand displacement amplification (SDA) for AFLP analysis: Genotyping single spores of arbuscular mycorrhizal (AM) fungi. J Microbiol Methods 63:157–164CrossRefPubMedGoogle Scholar
  20. 20.
    20. Dickson PA, Montgomery GW, Henders A, Campbell MJ, Martin NG, James MR (2005) Evaluation of multiple displacement amplification in a 5 cM STR genome-wide scan. Nucleic Acids Res. 33:ell9CrossRefGoogle Scholar
  21. 21.
    21. Pask R, Rance HE, Barratt BJ, Nutland S, Smyth DJ, Sebastian M, Twells RC, Smith A, Lam AC, Smink LJ et al (2004) Investigating the utility of combining phi29 whole genome amplification and highly multiplexed single nucleotide polymorphism BeadArray genotyping. BMC Biotechnol 4:15CrossRefPubMedGoogle Scholar
  22. 22.
    22. Lovmar L, Fredriksson M, Liljedahl U, Sigurdsson, S, Syvanen AC (2003) Quantitative evaluation by minisequencing and microarrays reveals accurate multiplexed SNP genotyping of whole genome amplified DNA. Nucleic Acids Res 31:e129CrossRefPubMedGoogle Scholar
  23. 23.
    23. Paez JG, Lin M, Beroukhim R, Lee JC, Zhao X, Richter DJ, Gabriel S, Herman P, Sasaki H, Altshuler D et al (2004) Genome coverage and sequence fidelity of phi29 polymerase-based multiple strand displacement whole genome amplification. Nucleic Acids Res 32:e71CrossRefPubMedGoogle Scholar
  24. 24.
    24. Montgomery GW, Campbell MJ, Dickson P, Herbert S, Siemering K, Ewen-White KR, Visscher PM, Martin NG (2005) Estimation of the rate of SNP genotyping errors from DNA extracted from different tissues. Twin Res Hum Genet 8:346–352CrossRefPubMedGoogle Scholar
  25. 25.
    25. Tzvetkov MV, Becker C, Kulle B, Nurnberg P, Brockmoller J, Wojnowski L (2005) Genome-wide single-nucleotide polymorphism arrays demonstrate high fidelity of multiple displacement-based whole-genome amplification. Electrophoresis 26:710–715CrossRefPubMedGoogle Scholar
  26. 26.
    26. Zhou X, Temam S, Chen Z, Ye H, Mao L, Wong DT (2005) Allelic imbalance analysis of oral tongue squamous cell carcinoma by high-density single nucleotide polymorphism arrays using whole-genome amplified DNA. Hum Genet 118:504–507CrossRefPubMedGoogle Scholar
  27. 27.
    27. Mai M, Hoyer JD, McClure RF (2004) Use of multiple displacement amplification to amplify genomic DNA before sequencing of the alpha and beta haemoglobin genes. J Clin Pathol 57:637–640CrossRefPubMedGoogle Scholar
  28. 28.
    28. Bergen AW, Qi Y, Haque KA, Welch RA, Chanock SJ (2005) Effects of DNA mass on multiple displacement whole genome amplification and genotyping performance. BMC Biotechnol 5:24CrossRefPubMedGoogle Scholar
  29. 29.
    29. Alanne M, Salomaa V, Saarela J, Peltonen L, Perola M (2004) DNA extraction yield is associated with several phenotypic characteristics: Results from two large population surveys. J. Thromb Haemost 2:2069–2071CrossRefPubMedGoogle Scholar
  30. 30.
    30. Gretarsdottir S, Thorleifsson G, Reynisdottir ST, Manolescu A, Jonsdottir S, Jonsdottir T, Gudmundsdottir T, Bjarnadottir SM, Einarsson OB, Gudjonsdottir HM et al (2003) The gene encoding phosphodiesterase 4D confers risk of ischemic stroke. Nat. Genet. 35:131–138CrossRefPubMedGoogle Scholar
  31. 31.
    31. Holbrook JF, Stabley D, Sol-Church K (2005) Exploring whole genome amplification as a DNA recovery tool for molecular genetic studies. J Biomol Tech 16:125–133PubMedGoogle Scholar
  32. 32.
    32. Jiang Z, Zhang X, Deka R, Jin L (2005) Genome amplification of single sperm using multiple displacement amplification. Nucleic Acids Res 33:e91CrossRefPubMedGoogle Scholar
  33. 33.
    33. Luthra R, Medeiros LJ (2004) Isothermal multiple displacement amplification: A highly reliable approach for generating unlimited high molecular weight genomic DNA from clinical specimens. J Mol Diagn. 6:236–242CrossRefPubMedGoogle Scholar
  34. 34.
    34. Silander K, Komulainen K, Ellonen P, Jussila M, Alanne M, Levander M, Tainola P, Kuulasmaa K, Salomaa V, Perola M et al (2005) Evaluating whole genome amplification via multiply-primed rolling circle amplification for SNP genotyping of samples with low DNA yield. Twin Res Hum Genet 8:368–375CrossRefPubMedGoogle Scholar
  35. 35.
    35. Dean FB, Nelson JR, Giesler TL, Lasken RS (2001) Rapid amplification of plasmid and phage DNA using Phi 29 DNA polymerase and multiply-primed rolling circle amplification. Genome Res 11:1095–1099CrossRefPubMedGoogle Scholar
  36. 36.
    36. Barker DL, Hansen MS, Faruqi AF, Giannola D, Irsula OR, Lasken RS, Latterich M, Makarov V, Oliphant A, Pinter JH et al (2004) Two methods of whole-genome amplification enable accurate genotyping across a 2320-SNP linkage panel. Genome Res 14:901–907CrossRefPubMedGoogle Scholar
  37. 37.
    37. Ng G, Roberts I, Coleman N (2005) Evaluation of three methods of whole-genome amplification for subsequent metaphase comparative genomic hybridization. Diagn Mol Pathol 14:203–212CrossRefPubMedGoogle Scholar
  38. 38.
    38. Sorensen KJ, Turteltaub K, Vrankovich G, Williams J, Christian AT (2004) Whole-genome amplification of DNA from residual cells left by incidental contact. Anal. Biochem. 324:312–314CrossRefPubMedGoogle Scholar
  39. 39.
    39. Thompson MD, Bowen, RA, Wong BY, Antal J, Liu Z, Yu H, Siminovitch K, Kreiger N, Rohan TE, Cole DE (2005) Whole genome amplification of buccal cell DNA: Genotyping concordance before and after multiple displacement amplification. Clin Chem Lab Med 43:157–162CrossRefPubMedGoogle Scholar
  40. 40.
    40. Handyside AH, Robinson MD, Simpson RJ, Omar MB, Shaw MA, Grudzinskas JG, Rutherford A (2004) Isothermal whole genome amplification from single and small numbers of cells: A new era for preimplantation genetic diagnosis of inherited disease. Mol Hum Reprod 10:767–772CrossRefPubMedGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2008

Authors and Affiliations

  • Kaisa Silander
    • 1
  • Janna Saarela
    • 1
  1. 1.National Public Health InstituteHelsinkiFinland

Personalised recommendations