Quantitative DNA Methylation Analysis at Single-Nucleotide Resolution by Pyrosequencing®.

Many protocols for gene-specific DNA methylation analysis are either labor intensive, not quantitative and/or limited to the measurement of the methylation status of only one or very few CpG positions. Pyrosequencing is a real-time sequencing technology that overcomes these limitations. After bisulfite modification of genomic DNA, a region of interest is amplified by PCR with one of the two primers being biotinylated.

Specific hypomethylated CpGs at the IGF2 locus act as an epigenetic biomarker for familial adenomatous polyposis colorectal cancer.

Aims: The identification of specific biomarkers for colorectal cancer is of primary importance for early diagnosis. The aim of this study was to evaluate if methylation changes at the IGF2/H19 locus could be predictive for individuals at high risk for developing sporadic or hereditary colorectal cancer.

Materials & Methods: Quantitative methylation analysis using pyrosequencing was performed on three differentially methylated regions (DMRs): IGF2 DMR0 and DMR2 and the H19 DMR in DNA samples from sporadic colorectal cancer (n = 26), familial adenomatous polyposis (n = 35) and hereditary nonpolyposis colorectal cancer (n = 19) patients.

Specific epigenetic alterations of IGF2-H19 locus in spermatozoa from infertile men.

DNA methylation marks, a key modification of imprinting, are erased in primordial germ cells and sex specifically re-established during gametogenesis. Abnormal epigenetic programming has been proposed as a possible mechanism compromising male fertility. We analysed by pyrosequencing the DNA methylation status of 47 CpGs located in differentially methylated regions (DMRs), the DMR0 and DMR2 of the IGF2 gene and in the 3rd and 6th CTCF-binding sites of the H19 DMR in human sperm from men with normal semen and patients with teratozoospermia (T) and/or oligo-astheno-teratozoospermia (OAT).

Identification of regions correlating MGMT promoter methylation and gene expression in glioblastomas.

The O(6)-methylguanine-DNA methyltransferase gene (MGMT) is methylated in several cancers, including gliomas. However, the functional role of cysteine-phosphate-guanine (CpG) island (CGI) methylation in MGMT silencing is still controversial. The aim of this study was to investigate whether MGMT CGI methylation correlates inversely with RNA expression of MGMT in glioblastomas and to determine the CpG region whose methylation best reflects the level of expression.

Identification and quantification of differentially methylated loci by the pyrosequencing technology.

Most available protocols for gene-specific DNA methylation analysis are either labor intensive, not quantitative, or limited to the measurement of the methylation status of only one or very few CpG positions. Pyrosequencing is a real-time sequencing technology that overcomes these limitations. After bisulfite modification of genomic DNA, a region of interest is amplified by polymerase chain reaction (PCR) with one of the two primers being biotinylated.