Skip to main content
View expanded cover

Whole Genome Amplification pp 69–86Cite as

Deterministic Whole-Genome Amplification of Single Cells

  • Protocol

Part of the Methods in Molecular Biology book series (MIMB,volume 1347)

Abstract

This chapter describes a single-cell whole genome amplification method (WGA) that has been originally published under the name “Single Cell Comparative Genomic Hybridization (SCOMP)” (Klein et al., Proc Natl Acad Sci U S A 96(8):4494–4499, 1999). The method has recently become available commercially under the name “Ampli1™ WGA Kit.” It is a PCR-based technique for whole genome amplification (WGA) allowing comprehensive and quite uniform amplification of DNA from low quantities of input DNA material, in particular single cells. The method is based on a ligation-mediated adaptor linker PCR approach. In contrast to other PCR-based WGA approaches, both the primer design and mechanism underlying the fragmentation of genome are nonrandom, enabling high priming efficiency and deterministic fragmentation of template DNA. This is particularly important for the design of (diagnostic) assays targeting specific loci. Here, we describe the WGA protocol for amplification of single-cell genomes designed to provide high-quality material in quantity sufficient for a number of locus-specific and genome-wide downstream assays [e.g., targeted Sanger sequencing, restriction fragment length polymorphism (RFLP), quantitative PCR (qPCR), and array comparative genomic hybridization (CGH)].

Key words

  • Whole genome amplification (WGA)
  • Single-cell analysis
  • Multiple displacement amplification (MDA)
  • Ligation-mediated PCR

This is a preview of subscription content, access via your institution.

Protocol
USD   49.95
Price excludes VAT (USA)
  • DOI: 10.1007/978-1-4939-2990-0_5
  • Chapter length: 18 pages
  • Instant PDF download
  • Readable on all devices
  • Own it forever
  • Exclusive offer for individuals only
  • Tax calculation will be finalised during checkout
eBook
USD   89.00
Price excludes VAT (USA)
  • ISBN: 978-1-4939-2990-0
  • Instant PDF download
  • Readable on all devices
  • Own it forever
  • Exclusive offer for individuals only
  • Tax calculation will be finalised during checkout
Softcover Book
USD   119.99
Price excludes VAT (USA)
Hardcover Book
USD   169.99
Price excludes VAT (USA)
Learn about institutional subscriptions
Fig. 1
Fig. 2

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. 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 U S A 96(8):4494–4499

    PubMed Central  CAS  CrossRef  PubMed  Google Scholar 

  2. 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(3):718–725

    CAS  CrossRef  PubMed  Google Scholar 

  3. 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(2)237–244. doi:10.1006/abio.2001.5460

    CAS  CrossRef  PubMed  Google Scholar 

  4. 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 U S A 89(13):5847–5851

    PubMed Central  CAS  CrossRef  PubMed  Google Scholar 

  5. Dietmaier W, Hartmann A, Wallinger S, Heinmoller E, Kerner T, Endl E, Jauch KW, Hofstadter F, Ruschoff J (1999) Multiple mutation analyses in single tumor cells with improved whole genome amplification. Am J Pathol 154(1):83–95. doi:10.1016/S0002-9440(10)65254-6

    PubMed Central  CAS  CrossRef  PubMed  Google Scholar 

  6. Kamberov E, Sun T, Bruening E, Pinter J, Sleptsova I, Kurihara T, Makarov V (2004) Amplification and analysis of whole genome and whole transcriptome libraries generated by a DNA polymerization process. United States Patent Application Publication no. US 2004/0209298 A1

    Google Scholar 

  7. Zong C, Lu S, Chapman AR, Xie XS (2012) Genome-wide detection of single-nucleotide and copy-number variations of a single human cell. Science 338(6114):1622–1626. doi:10.1126/science.1229164

    PubMed Central  CAS  CrossRef  PubMed  Google Scholar 

  8. Langmore JP (2002) Rubicon Genomics, Inc. Pharmacogenomics 3(4):557–560. doi:10.1517/14622416.3.4.557

    CrossRef  PubMed  Google Scholar 

  9. Tanabe C, Aoyagi K, Sakiyama T, Kohno T, Yanagitani N, Akimoto S, Sakamoto M, Sakamoto H, Yokota J, Ohki M, Terada M, Yoshida T, Sasaki H (2003) Evaluation of a whole-genome amplification method based on adaptor-ligation PCR of randomly sheared genomic DNA. Genes Chromosomes Cancer 38(2):168–176. doi:10.1002/gcc.10269

    CAS  CrossRef  PubMed  Google Scholar 

  10. Schardt JA, Meyer M, Hartmann CH, Schubert F, Schmidt-Kittler O, Fuhrmann C, Polzer B, Petronio M, Eils R, Klein CA (2005) Genomic analysis of single cytokeratin-positive cells from bone marrow reveals early mutational events in breast cancer. Cancer Cell 8(3):227–239. doi:10.1016/j.ccr.2005.08.003

    CAS  CrossRef  PubMed  Google Scholar 

  11. Stoecklein NH, Hosch SB, Bezler M, Stern F, Hartmann CH, Vay C, Siegmund A, Scheunemann P, Schurr P, Knoefel WT, Verde PE, Reichelt U, Erbersdobler A, Grau R, Ullrich A, Izbicki JR, Klein CA (2008) Direct genetic analysis of single disseminated cancer cells for prediction of outcome and therapy selection in esophageal cancer. Cancer Cell 13(5):441–453. doi:10.1016/j.ccr.2008.04.005

    CAS  CrossRef  PubMed  Google Scholar 

  12. Klein CA, Blankenstein TJ, Schmidt-Kittler O, Petronio M, Polzer B, Stoecklein NH, Riethmuller G (2002) Genetic heterogeneity of single disseminated tumour cells in minimal residual cancer. Lancet 360(9334):683–689. doi:10.1016/S0140-6736(02)09838-0

    CAS  CrossRef  PubMed  Google Scholar 

  13. Schmidt-Kittler O, Ragg T, Daskalakis A, Granzow M, Ahr A, Blankenstein TJ, Kaufmann M, Diebold J, Arnholdt H, Muller P, Bischoff J, Harich D, Schlimok G, Riethmuller G, Eils R, Klein CA (2003) From latent disseminated cells to overt metastasis: genetic analysis of systemic breast cancer progression. Proc Natl Acad Sci U S A 100(13):7737–7742. doi:10.1073/pnas.1331931100

    PubMed Central  CAS  CrossRef  PubMed  Google Scholar 

  14. Ulmer A, Schmidt-Kittler O, Fischer J, Ellwanger U, Rassner G, Riethmuller G, Fierlbeck G, Klein CA (2004) Immunomagnetic enrichment, genomic characterization, and prognostic impact of circulating melanoma cells. Clin Cancer Res 10(2):531–537

    CAS  CrossRef  PubMed  Google Scholar 

  15. Ulmer A, Dietz K, Hodak I, Polzer B, Scheitler S, Yildiz M, Czyz Z, Lehnert P, Fehm T, Hafner C, Schanz S, Rocken M, Garbe C, Breuninger H, Fierlbeck G, Klein CA (2014) Quantitative measurement of melanoma spread in sentinel lymph nodes and survival. PLoS Med 11(2), e1001604. doi:10.1371/journal.pmed.1001604

    PubMed Central  CrossRef  PubMed  Google Scholar 

  16. Weckermann D, Polzer B, Ragg T, Blana A, Schlimok G, Arnholdt H, Bertz S, Harzmann R, Klein CA (2009) Perioperative activation of disseminated tumor cells in bone marrow of patients with prostate cancer. J Clin Oncol 27(10):1549–1556. doi:10.1200/JCO.2008.17.0563

    CrossRef  PubMed  Google Scholar 

  17. Fuhrmann C, Schmidt-Kittler O, Stoecklein NH, Petat-Dutter K, Vay C, Bockler K, Reinhardt R, Ragg T, Klein CA (2008) High-resolution array comparative genomic hybridization of single micrometastatic tumor cells. Nucleic Acids Res 36(7), e39. doi:10.1093/nar/gkn101

    PubMed Central  CrossRef  PubMed  Google Scholar 

  18. Czyz ZT, Hoffmann M, Schlimok G, Polzer B, Klein CA (2014) Reliable single cell array CGH for clinical samples. PLoS One 9(1), e85907. doi:10.1371/journal.pone.0085907

    PubMed Central  CrossRef  PubMed  Google Scholar 

  19. Mohlendick B, Bartenhagen C, Behrens B, Honisch E, Raba K, Knoefel WT, Stoecklein NH (2013) A robust method to analyze copy number alterations of less than 100 kb in single cells using oligonucleotide array CGH. PLoS One 8(6):e67031. doi:10.1371/journal.pone.0067031

    PubMed Central  CrossRef  PubMed  Google Scholar 

  20. Polzer B, Medoro G, Pasch S, Fontana F, Zorzino L, Pestka A, Andergassen U, Meier-Stiegen F, Czyz ZT, Alberter B, Treitschke S, Schamberger T, Sergio M, Bregola G, Doffini A, Gianni S, Calanca A, Signorini G, Bolognesi C, Hartmann A, Fasching PA, Sandri MT, Rack B, Fehm T, Giorgini G, Manaresi N, Klein CA (2014) Molecular profiling of single circulating tumor cells with diagnostic intention. EMBO Mol Med 6(11):1371–1386. doi:10.15252/emmm.201404033

    PubMed Central  CAS  CrossRef  PubMed  Google Scholar 

  21. Stoecklein NH, Erbersdobler A, Schmidt-Kittler O, Diebold J, Schardt JA, Izbicki JR, Klein CA (2002) SCOMP is superior to degenerated oligonucleotide primed-polymerase chain reaction for global amplification of minute amounts of DNA from microdissected archival tissue samples. Am J Pathol 161(1):43–51

    PubMed Central  CrossRef  PubMed  Google Scholar 

  22. Arneson N, Moreno J, Iakovlev V, Ghazani A, Warren K, McCready D, Jurisica I, Done SJ (2012) Comparison of whole genome amplification methods for analysis of DNA extracted from microdissected early breast lesions in formalin-fixed paraffin-embedded tissue. ISRN Oncol 2012:710692. doi:10.5402/2012/710692

    PubMed Central  PubMed  Google Scholar 

  23. Binder V, Bartenhagen C, Okpanyi V, Gombert M, Moehlendick B, Behrens B, Klein HU, Rieder H, Ida Krell PF, Dugas M, Stoecklein NH, Borkhardt A (2014) A new workflow for whole-genome sequencing of single human cells. Hum Mutat 35:1260–1270. doi:10.1002/humu.22625

    CAS  CrossRef  PubMed  Google Scholar 

  24. Czyz ZT (2014) Development of a reliable single-cell aCGH suitable for clinical samples. Dissertation, University of Regensburg. urn:nbn:de:bvb:355-epub-318818, http://epub.uni-regensburg.de/31881/

Download references

Author information

Authors and Affiliations

  1. Project Group, Personalized Tumor Therapy, Fraunhofer Institute for Toxicology and Experimental Medicine (ITEM), Regensburg, Germany

    Zbigniew Tadeusz Czyż & Christoph A. Klein

  2. Experimental Medicine and Therapy Research, University of Regensburg, Regensburg, Germany

    Christoph A. Klein

Authors
  1. Zbigniew Tadeusz Czyż
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Christoph A. Klein
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Christoph A. Klein .

Editor information

Editors and Affiliations

  1. Institute of Cell Biology, Histology & Embryology, Medical University of Graz, Graz, Austria

    Thomas Kroneis

Rights and permissions

Reprints and Permissions

Copyright information

© 2015 Springer Science+Business Media New York

About this protocol

Cite this protocol

Czyż, Z.T., Klein, C.A. (2015). Deterministic Whole-Genome Amplification of Single Cells. In: Kroneis, T. (eds) Whole Genome Amplification. Methods in Molecular Biology, vol 1347. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2990-0_5

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-2990-0_5

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-2989-4

  • Online ISBN: 978-1-4939-2990-0

  • eBook Packages: Springer Protocols