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Developing Noninvasive Diagnosis for Single-Gene Disorders: The Role of Digital PCR

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Quantitative Real-Time PCR

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1160))

Abstract

Cell-free fetal DNA constitutes approximately 10 % of the cell-free DNA found in maternal plasma and can be used as a reliable source of fetal genetic material for noninvasive prenatal diagnosis (NIPD) from early pregnancy. The relatively high levels of maternal background can make detection of paternally inherited point mutations challenging. Diagnosis of inheritance of autosomal recessive disorders using qPCR is even more challenging due to the high background of mutant maternal allele. Digital PCR is a very sensitive modified method of quantitative real-time PCR (qPCR), allowing absolute quantitation and rare allele detection without the need for standards or normalization. Samples are diluted and then partitioned into a large number of small qPCR reactions, some of which contain the target molecule and some which do not; the proportion of positive reactions can be used to calculate the concentration of targets in the initial sample. Here we discuss the use of digital PCR as an accurate approach to NIPD for single-gene disorders.

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Author information

Authors and Affiliations

  1. Department of Obstetrics and Gynaecology, Yong Loo Lin School of Medicine, National University of Singapore, NUHS Tower Block, Level 12, 1E Kent Ridge Road, Singapore, 119228, Singapore

    Angela N. Barrett

  2. Clinical and Molecular Genetics Unit, UCL Institute of Child Health, London, UK

    Lyn S. Chitty

  3. North East Thames Regional Genetics Laboratory, Great Ormond Street Hospital, London, UK

    Lyn S. Chitty

Authors
  1. Angela N. Barrett
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  2. Lyn S. Chitty
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Corresponding author

Correspondence to Angela N. Barrett .

Editor information

Editors and Affiliations

  1. Laboratorio Medicina Molecolare, Dipartimento Medicina Traslazionale, Istituto Giannina Gaslini, Genova, Italy

    Roberto Biassoni

  2. Laboratorio U.O.C Neurochirurgia, Istituto Giannina Gaslini, Genova, Italy

    Alessandro Raso

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© 2014 Springer Science+Business Media New York

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Barrett, A.N., Chitty, L.S. (2014). Developing Noninvasive Diagnosis for Single-Gene Disorders: The Role of Digital PCR. In: Biassoni, R., Raso, A. (eds) Quantitative Real-Time PCR. Methods in Molecular Biology, vol 1160. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-0733-5_17

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  • DOI: https://doi.org/10.1007/978-1-4939-0733-5_17

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-0732-8

  • Online ISBN: 978-1-4939-0733-5

  • eBook Packages: Springer Protocols

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