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.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Stankiewicz P, Lupski JR (2010) Structural variation in the human genome and its role in disease. Annu Rev Med 61:437–455
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 22:476–485
Owen RP, Sangkuhl K, Klein TE et al (2009) Cytochrome P450 2D6. Pharmacogenet Genomics 19:559–562
Soderback E, Zackrisson AL, Lindblom B et al (2005) Determination of CYP2D6 gene copy number by pyrosequencing. Clin Chem 51:522–531
Pielberg G, Day AE, Plastow GS et al (2003) A sensitive method for detecting variation in copy numbers of duplicated genes. Genome Res 13:2171–2177
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 5:425
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 19:2081–2089
Liu Z, Schneider DL, Kornfeld K et al (2010) Simple copy number determination with reference query pyrosequencing (RQPS). Cold Spring Harb Protoc 9:5491
Setty P, Gessi M, Waha A et al (2011) Sensitive determination of BRAF copy number in clinical samples by pyrosequencing. Diagn Mol Pathol 20:148–157
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2015 Springer Science+Business Media New York
About this protocol
Cite this protocol
Kringen, M.K. (2015). Analysis of Copy Number Variation by Pyrosequencing® Using Paralogous Sequences. In: Lehmann, U., Tost, J. (eds) Pyrosequencing. Methods in Molecular Biology, vol 1315. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2715-9_9
Download citation
DOI: https://doi.org/10.1007/978-1-4939-2715-9_9
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-2714-2
Online ISBN: 978-1-4939-2715-9
eBook Packages: Springer Protocols