An analytical method for the detection of methylation differences at specific chromosomal loci using primer extension and ion pair reverse phase HPLC

  title={An analytical method for the detection of methylation differences at specific chromosomal loci using primer extension and ion pair reverse phase HPLC},
  author={Maryam M. Matin and Alessandra Baumer and David P Hornby},
  journal={Human Mutation},
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… 

Precision and performance characteristics of bisulfite conversion and real-time PCR (MethyLight) for quantitative DNA methylation analysis.

Assays to measure DNA methylation, which are important in epigenetic research and clinical diagnostics, typically rely on conversion of unmethylated cytosine to uracil by sodium bisulfite. However,

Detection of genomic DNA methylation with denaturing high performance liquid chromatography

DHPLC technology serves as a rapid screening tool to monitor the genomic DNA methylation and could be used to increase the throughput efficiency of methylation analysis.

Methylation-Specific PCR Unraveled

This review discusses the critical parameters of MSP and presents an overview of the available MSP variants and the (clinical) applications.

CpG methylation analysis--current status of clinical assays and potential applications in molecular diagnostics: a report of the Association for Molecular Pathology.

The Methylation Working Group of the Clinical Practice Committee of the Association of Molecular Pathology has reviewed the current state of clinical testing in this area and reports a summary of both the advantages and disadvantages of various methods, as well as the needs for standardization and reporting.

Nucleic Acid Chromatography

The intent of the authors that this article should help molecular biologists how to use DNA chromatography effectively in their research.

Colorectal Cancer Epigenetics: The Role of Environmental Factors and the Search for Molecular Biomarkers

  • F. Ahmed
  • Biology
    Journal of environmental science and health. Part C, Environmental carcinogenesis & ecotoxicology reviews
  • 2007
An evenhanded evaluation of the role of epigenetics in the development of colorectal cancer is presented, and the extent of environmental influences on modulating this disease is investigated.

Epigenetic changes in virus-associated human cancers

This review will focus on hypermethylation and tumor suppressor gene silencing and the signal pathways that are involved, particularly in cancers closely associated with the hepatitis B virus, simian virus 40 (SV40), and Epstein-Barr virus.

Early detection: The power and the promise of DNA methylation markers

  • P. Laird
  • Biology
    Nature Reviews Cancer
  • 2003
The past few years have seen an explosion of interest in the epigenetics of cancer. This has been a consequence of both the exciting coalescence of the chromatin and DNA methylation fields, and the



A novel MSP/DHPLC method for the investigation of the methylation status of imprinted genes enables the molecular detection of low cell mosaicisms

The MSP/DHPLC method offers a rapid and very reliable alternative to conventional methods used for such purposes as Southern blots and methylation specific PCR (allele‐specific MSP), and may also reveal mosaicisms concerning structurally intact chromosomes.

Rapid quantitation of methylation differences at specific sites using methylation-sensitive single nucleotide primer extension (Ms-SNuPE).

This methylation-sensitive technique has several advantages over existing methods used for detection of methylation changes because small amounts of DNA can be analyzed including microdissected pathology sections and it avoids utilization of restriction enzymes for determining the methylation status at CpG sites.

Cytosine methylation: quantitation by automated genomic sequencing and GENESCAN analysis.

An accurate and innovative protocol to directly quantitate the methylation of any cytosine residue in the target sequence by fluorescence-based automated genomic sequencing is described and the unexpected result that multicopy plasmid DNA grown in a Dcm host is not always fully methylated is found.

High sensitivity mapping of methylated cytosines.

A genomic sequencing technique which is capable of detecting every methylated cytosine on both strands of any target sequence, using DNA isolated from fewer than 100 cells is developed.

A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands.

A genomic sequencing method is reported that provides positive identification of 5-methylcytosine residues and yields strand-specific sequences of individual molecules in genomic DNA, which suggests that the high methylation level of single-copy sequences in sperm may be locally modulated by binding of protein factors in germ-line cells.

Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands.

The use of MSP is demonstrated to identify promoter region hypermethylation changes associated with transcriptional inactivation in four important tumor suppressor genes (p16, p15, E-cadherin and von Hippel-Lindau) in human cancer.

A modified and improved method for bisulphite based cytosine methylation analysis.

An improved modification of the bisulphite based sequencing method is presented that facilitates the handling of probes and reproducibly reaches a very high level of sensitivity.

Genotyping single nucleotide polymorphisms by primer extension and high performance liquid chromatography

It is shown that robust signal is obtained using this simple analytic method which has the added advantages that sample loading and analysis are essentially automated, analytic time is brief, and no further purification step after primer extension is required.

Denaturing high‐performance liquid chromatography: A review

The utility of DHPLC has been extended to the genotyping of known polymorphisms by utilizing the ability of poly(styrene‐divinylbenzene) to resolve single‐stranded DNA molecules of identical size that differ in a single base.

Use of a HpaII-polymerase chain reaction assay to study DNA methylation in the Pgk-1 CpG island of mouse embryos at the time of X-chromosome inactivation

It was found that HpaII site H-7 in the CpG island of the X-chromosome-linked Pgk-1 gene is less than or equal to 10% methylated in oocytes and male embryos but becomes 40%methylated in female embryos at 6.5 days; about the time of X- chromosome inactivation of the inner cell mass.