Abstract
Precise quantitation of allelic burden for a pathogenic mutation has diverse clinical and research applications but can be difficult to achieve with conventional qPCR-based techniques, especially at lower mutant allele frequencies. Digital PCR overcomes many of the limitations of qPCR and can be highly quantitative even for single-nucleotide variants, with distinct advantages over next-generation sequencing approaches. Here we describe a method combining the principles of TaqMan®-chemistry SNP genotyping with microfluidic digital PCR to generate a highly sensitive, quantitative allele-specific digital PCR assay for the six most common IDH1 and IDH2 mutations encountered in myeloid malignancy. The concept and approach could easily be applied to other suitable SNVs.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsSpringer Nature is developing a new tool to find and evaluate Protocols. Learn more
References
Losman JA, Kaelin WG (2013) What a difference a hydroxyl makes: mutant IDH, (R)-2-hydroxyglutarate, and cancer. Genes Dev 27:836–852
Thiede C, Steudel C, Mohr B et al (2002) Analysis of FLT3-activating mutations in 979 patients with acute myelogenous leukemia: association with FAB subtypes and identification of subgroups with poor prognosis. Blood 99:4326–4335
Grimwade D, Freeman SD (2014) Defining minimal residual disease in acute myeloid leukemia: which platforms are ready for “prime time”? Blood 124:3345–3355
Schnittger S, Kern W, Tschulik C et al (2009) Minimal residual disease levels assessed by NPM1 mutation-specific RQ-PCR provide important prognostic information in AML. Blood 114:2220–2231
Roma C, Esposito C, Rachiglio AM et al (2013) Detection of EGFR mutations by TaqMan mutation detection assays powered by competitive allele-specific TaqMan PCR technology. Biomed Res Int 2013:385087
Matsuda K, Sugano M, Honda T (2012) PCR for monitoring of minimal residual disease in hematologic malignancy. Clin Chim Acta 413:74–80
Huggett JF, Foy CA, Benes V et al (2013) The digital MIQE guidelines: minimum information for publication of quantitative digital PCR experiments. Clin Chem 59:892–902
Vogelstein B, Kinzler KW (1999) Digital PCR. Proc Natl Acad Sci U S A 96:9236–9241
Dube S, Qin J, Ramakrishnan R (2008) Mathematical analysis of copy number variation in a DNA sample using digital PCR on a nanofluidic device. PLoS One 3:e2876
Bhat S, Herrmann J, Armishaw P et al (2009) Single molecule detection in nanofluidic digital array enables accurate measurement of DNA copy number. Anal Bioanal Chem 394:457–467
Huggett JF, Cowen S, Foy CA (2015) Considerations for digital PCR as an accurate molecular diagnostic tool. Clin Chem 61:79–88
Wiseman DH, Struys EA, Wilks DP et al (2015) Direct comparison of quantitative digital PCR and 2-hydroxyglutarate enantiomeric ratio for IDH mutant allele frequency assessment in myeloid malignancy. Leukemia 29:2421–2423
Shen GQ, Abdullah KG, Wang QK (2009) The TaqMan method for SNP genotyping. Methods Mol Biol 578:293–306
Schleinitz D, Distefano JK, Kovacs P (2011) Targeted SNP genotyping using the TaqMan® assay. Methods Mol Biol 700:77–87
Whale AS, Cowen S, Foy CA et al (2013) Methods for applying accurate digital PCR analysis on low copy DNA samples. PLoS One 8:e58177
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2017 Springer Science+Business Media LLC
About this protocol
Cite this protocol
Wiseman, D.H., Somervaille, T.C.P. (2017). Nanofluidic Allele-Specific Digital PCR Method for Quantifying IDH1 and IDH2 Mutation Burden in Acute Myeloid Leukemia. In: Fortina, P., Londin, E., Park, J., Kricka, L. (eds) Acute Myeloid Leukemia. Methods in Molecular Biology, vol 1633. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7142-8_15
Download citation
DOI: https://doi.org/10.1007/978-1-4939-7142-8_15
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-7140-4
Online ISBN: 978-1-4939-7142-8
eBook Packages: Springer Protocols