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Pyrosequencing pp 241-258 | Cite as

Detection of Loss of Imprinting by Pyrosequencing®

  • Silvia Tabano
  • Eleonora Bonaparte
  • Monica MiozzoEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1315)

Abstract

Genomic imprinting is an epigenetically regulated process determining allele-specific expression in a parent-of-origin dependent manner. Altered expression of imprinted genes characterizes numerous congenital diseases including Beckwith–Wiedemann, Silver–Russell, Angelman, and Prader–Willi syndromes as well as acquired disorders such as cancer. The detection of imprinting alterations has important translational implications in clinics and the application of the Pyrosequencing® technology offers the possibility to identify accurately also subtle modifications in allele-specific expression and in DNA methylation levels.

Here, we describe two methods to investigate genomic imprinting defects (loss of imprinting, LOI) using Pyrosequencing: (1) Allele-specific expression analysis based on single nucleotide polymorphism (SNP), and (2) quantification of DNA methylation.

The protocol for the quantification of the allele-specific expression is carried out by analyzing an informative SNP located within the transcribed portion of an imprinted gene. The method includes the cDNA amplification of the region containing the SNP and the Pyrosequencing-based analysis for the quantitative allelic discrimination comparing the ratio of the two alleles.

The second protocol allows the accurate quantification of the DNA methylation levels at the Imprinting Control Regions (ICRs). Imprinted genes are clustered in chromosomal regions and their expression is mainly regulated by DNA methylation at CpG sites located within the ICRs. After bisulfite modification of the genomic DNA, the region of interest is amplified by PCR and analyzed by Pyrosequencing. The methylation value at each CpG site is calculated by the CpG software, which determines the ratio of the incorporation of “C” and “T” and converts the value in methylation percentage.

Key words

Genomic imprinting Epigenetics DNA methylation Pyrosequencing® Allele-specific expression Loss of imprinting ICR Prader–Willi syndrome Angelman syndrome Beckwith–Wiedemann syndrome Silver–Russell syndrome 

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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Silvia Tabano
    • 1
    • 2
  • Eleonora Bonaparte
    • 2
  • Monica Miozzo
    • 1
    • 2
    Email author
  1. 1.Department of Pathophysiology and TransplantationUniversità degli Studi di MilanoMilanItaly
  2. 2.Division of PathologyFondazione IRCCS Ca’ Granda Ospedale Maggiore PoliclinicoMilanItaly

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