Abstract
One of the major reasons for pregnant women to ask for prenatal diagnosis is to detect fetal chromosomal aneuploidies. Analysis of allele ratios of SNPs has been used for prenatal detection of fetal aneuploidies using MALDI-TOF mass spectrometry (MS). However, quantitative SNP genotyping by MALDI-TOF MS is challenging. To obtain a better quantification of allelic ratios, a Pyrosequencing® protocol for SNP genotyping has been developed to perform prenatal diagnosis of aneuploidies.
To avoid the laborious process and risk of cross-contamination brought in by DNA extraction procedures, a PCR assay, which can amplify DNA directly from cells in amniotic fluid, has been developed. Pre-amplification steps such as cell enrichment and heating are required to obtain sufficient amounts of amplification products.
In this chapter, SNPs on chromosome 21 are used to detect trisomy 21 as an example of aneuploidy by quantifying the allele ratio using Pyrosequencing. Primer selection for PCRs and Pyrosequencing reactions, optimization of nucleotide dispensation orders, establishment of cutoff values for trisomy 21, and interpretation of data are all factors essential for a successful diagnosis and are discussed in detail herein.
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Acknowledgments
This work was supported by the National Natural Science Foundation of China (project number: 21275161 and 21005088) and the National Key Basic Research Program of China (2014CB744501).
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Ye, H. et al. (2015). Prenatal Diagnosis of Chromosomal Aneuploidies by Quantitative Pyrosequencing® . 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_10
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DOI: https://doi.org/10.1007/978-1-4939-2715-9_10
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-2714-2
Online ISBN: 978-1-4939-2715-9
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