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Pyrosequencing pp 115-121 | Cite as

Analysis of Copy Number Variation by Pyrosequencing® Using Paralogous Sequences

  • Marianne Kristiansen Kringen
Part of the Methods in Molecular Biology book series (MIMB, volume 1315)

Abstract

The determination of gene copy numbers of highly similar genes is difficult with conventional PCR-based methods. However, by amplification of similar genes in the same PCR reaction followed by Pyrosequencing®, one may distinguish the genes based on a single-nucleotide difference. The ratio between the peak heights of gene-specific nucleotides obtained in the Pyrosequencing reaction may thereby be used to calculate the relative copy numbers of target genes. This method is easy and cost effective compared to other methods, and allows for the determination of copy numbers of genes that were previously difficult to achieve.

Key words

Copy number variation PCR Pyrosequencing® Paralogous sequences ABCC6 

References

  1. 1.
    Stankiewicz P, Lupski JR (2010) Structural variation in the human genome and its role in disease. Annu Rev Med 61:437–455PubMedCrossRefGoogle Scholar
  2. 2.
    Schaeffeler E, Schwab M, Eichelbaum M et al (2003) CYP2D6 genotyping strategy based on gene copy number determination by TaqMan real-time PCR. Hum Mutat 22:476–485PubMedCrossRefGoogle Scholar
  3. 3.
    Owen RP, Sangkuhl K, Klein TE et al (2009) Cytochrome P450 2D6. Pharmacogenet Genomics 19:559–562PubMedCentralPubMedCrossRefGoogle Scholar
  4. 4.
    Soderback E, Zackrisson AL, Lindblom B et al (2005) Determination of CYP2D6 gene copy number by pyrosequencing. Clin Chem 51:522–531PubMedCrossRefGoogle Scholar
  5. 5.
    Pielberg G, Day AE, Plastow GS et al (2003) A sensitive method for detecting variation in copy numbers of duplicated genes. Genome Res 13:2171–2177PubMedCentralPubMedCrossRefGoogle Scholar
  6. 6.
    Kringen MK, Stormo C, Grimholt RM et al (2012) Copy number variations of the ATP-binding cassette transporter ABCC6 gene and its pseudogenes. BMC Res Notes 5:425PubMedCentralPubMedCrossRefGoogle Scholar
  7. 7.
    Liu Z, Obenauf AC, Speicher MR et al (2009) Rapid identification of homologous recombinants and determination of gene copy number with reference/query pyrosequencing (RQPS). Genome Res 19:2081–2089PubMedCentralPubMedCrossRefGoogle Scholar
  8. 8.
    Liu Z, Schneider DL, Kornfeld K et al (2010) Simple copy number determination with reference query pyrosequencing (RQPS). Cold Spring Harb Protoc 9:5491Google Scholar
  9. 9.
    Setty P, Gessi M, Waha A et al (2011) Sensitive determination of BRAF copy number in clinical samples by pyrosequencing. Diagn Mol Pathol 20:148–157PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Department of PharmacologyOslo University Hospital, UllevålOsloNorway

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