A real-time PCR assay for DNA-methylation using methylation-specific blockers.
DNA methylation-based biomarkers have been discovered that could potentially be used for the diagnosis of cancer by detection of circulating, tumor-derived DNA in bodily fluids. Any methylation detection assay that would be applied to these samples must be capable of detecting small amounts of tumor DNA in the presence of background normal DNA. We have developed a real-time PCR assay, called HeavyMethyl, that is well suited for this application. HeavyMethyl uses methylation-specific oligonucleotide blockers and a methylation-specific probe to achieve methylation-specific amplification and detection. We tested the assays on unmethylated and artificially methylated DNA in order to determine the limit of detection. After careful optimization, our glutathione-S-transferase pi1 and Calcitonin assays can amplify as little as 30 and 60 pg of methylated DNA, respectively, and neither assay amplifies unmethylated DNA. The Calcitonin assay showed a highly significant methylation difference between normal colon and colon adenocarcinomas, and methylation was also detected in serum DNA from colon cancer patients. These assays show that HeavyMethyl technology can be successfully employed for the analysis of very low concentrations of methylated DNA, e.g. in serum of patients with tumors.
Cottrell
Susan E
SE
Epigenomics Inc, 1000 Seneca Street, Suite 300 Seattle, WA 98101, USA.
Distler
Jürgen
J
Goodman
Nancy S
NS
Mooney
Suzanne H
SH
Kluth
Antje
A
Olek
Alexander
A
Schwope
Ina
I
Tetzner
Reimo
R
Ziebarth
Heike
H
Berlin
Kurt
K
D000230:Adenocarcinoma / Q000097:blood / Q000175:diagnosis / Q000235:genetics; D001483:Base Sequence; D002116:Calcitonin / Q000235:genetics; D003110:Colonic Neoplasms / Q000097:blood / Q000175:diagnosis / Q000235:genetics; D004247:DNA / Q000032:analysis* / Q000235:genetics / Q000378:metabolism*; D019175:DNA Methylation; D017931:DNA Primers / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D051549:Glutathione S-Transferase pi; D005982:Glutathione Transferase / Q000235:genetics; D006801:Humans; D007527:Isoenzymes / Q000235:genetics; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000235:genetics / Q000378:metabolism*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013447:Sulfites / Q000378:metabolism; D013997:Time Factors
D017931:DNA Primers; D007527:Isoenzymes; D009841:Oligonucleotides; D013447:Sulfites; D002116:Calcitonin; D004247:DNA; C496556:GSTP1 protein, human; D051549:Glutathione S-Transferase pi; D005982:Glutathione Transferase; C042345:hydrogen sulfite