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An analytical method for the detection of methylation differences at specific chromosomal loci using primer extension and ion pair reverse phase HPLC
Author(s) -
Matin Maryam M.,
Baumer Alessandra,
Hornby David P.
Publication year - 2002
Publication title -
human mutation
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 162
eISSN - 1098-1004
pISSN - 1059-7794
DOI - 10.1002/humu.10118
Subject(s) - biology , primer (cosmetics) , primer extension , genetics , methylation , microbiology and biotechnology , computational biology , chromatography , dna , base sequence , chemistry , organic chemistry
We have developed a rapid, accurate, and quantitative method for the detection of methylation differences at specific CpG sites based on bisulfite treatment of DNA followed by primer extension and ion‐pair reversed‐phase high performance liquid chromatography (IP RP HPLC). The application of the method is illustrated by analysis of differentially imprinted alleles arising from Prader‐Willi and Angelman syndromes. In order to convert unmethylated cytosines to uracil, plasmid and genomic DNA samples were treated with sodium bisulfite and the targeted sequence was then amplified using oligodeoxynucleotide primers specific for the bisulfite‐deaminated DNA. The PCR product(s) from this step was used as a template for a primer extension reaction and the products were subsequently analyzed chromatographically using IP RP HPLC. This method eliminates the need to use restriction enzymes to determine the methylation status of the amplicon and circumvents the need for radio labeling for the quantitative measurements. Finally, this method removes the need for nucleotide sequencing because it is not solely reliant on the presence or absence of one or more PCR products, as is the case with related methods. Hum Mutat 20:305–311, 2002. © 2002 Wiley‐Liss, Inc.