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Analysis of the methylation status of imprinted genes based on methylation-specific polymerase chain reaction combined with denaturing high-performance liquid chromatography


Baumer Wolz, Alessandra (2002). Analysis of the methylation status of imprinted genes based on methylation-specific polymerase chain reaction combined with denaturing high-performance liquid chromatography. Methods, 27(2):139-43.

Abstract

A procedure for the analysis of the methylation status of imprinted genes is described. The method offers a rapid and reliable alternative to conventional methods such as Southern blots and methylation-specific polymerase chain reaction (PCR) (i.e., allele-specific methylation-specific PCR). The efficient resolution of the differentially methylated alleles is demonstrated for three human imprinted genes: SNRPN, LIT1 (alias KCNQ1OT1), and H19. Abnormal imprinting of SNRPN is associated with the Angelman/Prader-Willi syndromes, and that of LIT1 and H19 with the Beckwith-Wiedemann syndrome. The method is based on methylation-specific PCR followed by denaturing high-performance liquid chromatography (MSP/DHPLC). Briefly, genomic DNA is initially subjected to an in vitro bisulfite treatment, whereby unmethylated cytosines are deaminated. Subsequent PCR amplifications, using primers specific for modified DNA, are aimed at DNA segments that show parent-of-origin-specific methylation. PCR conditions are chosen that allow an efficient amplification of both alleles. The PCR products representing the two alleles are identical in size; they differ, however, at a number of positions within the amplified DNA segment. The DHPLC analysis allows very efficient resolution of the two populations of PCR products, providing qualitative and quantitative results.

Abstract

A procedure for the analysis of the methylation status of imprinted genes is described. The method offers a rapid and reliable alternative to conventional methods such as Southern blots and methylation-specific polymerase chain reaction (PCR) (i.e., allele-specific methylation-specific PCR). The efficient resolution of the differentially methylated alleles is demonstrated for three human imprinted genes: SNRPN, LIT1 (alias KCNQ1OT1), and H19. Abnormal imprinting of SNRPN is associated with the Angelman/Prader-Willi syndromes, and that of LIT1 and H19 with the Beckwith-Wiedemann syndrome. The method is based on methylation-specific PCR followed by denaturing high-performance liquid chromatography (MSP/DHPLC). Briefly, genomic DNA is initially subjected to an in vitro bisulfite treatment, whereby unmethylated cytosines are deaminated. Subsequent PCR amplifications, using primers specific for modified DNA, are aimed at DNA segments that show parent-of-origin-specific methylation. PCR conditions are chosen that allow an efficient amplification of both alleles. The PCR products representing the two alleles are identical in size; they differ, however, at a number of positions within the amplified DNA segment. The DHPLC analysis allows very efficient resolution of the two populations of PCR products, providing qualitative and quantitative results.

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Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > Institute of Medical Genetics
Dewey Decimal Classification:570 Life sciences; biology
610 Medicine & health
Scopus Subject Areas:Life Sciences > Molecular Biology
Life Sciences > General Biochemistry, Genetics and Molecular Biology
Language:English
Date:June 2002
Deposited On:26 May 2021 14:24
Last Modified:25 Apr 2024 01:37
Publisher:Elsevier
ISSN:1046-2023
OA Status:Closed
Publisher DOI:https://doi.org/10.1016/s1046-2023(02)00066-x
PubMed ID:12095272