Combination of methylated-DNA precipitation and methylation-sensitive restriction enzymes (COMPARE-MS) for the rapid, sensitive and quantitative detection of DNA methylation.
Hypermethylation of CpG island (CGI) sequences is a nearly universal somatic genome alteration in cancer. Rapid and sensitive detection of DNA hypermethylation would aid in cancer diagnosis and risk stratification. We present a novel technique, called COMPARE-MS, that can rapidly and quantitatively detect CGI hypermethylation with high sensitivity and specificity in hundreds of samples simultaneously. To quantitate CGI hypermethylation, COMPARE-MS uses real-time PCR of DNA that was first digested by methylation-sensitive restriction enzymes and then precipitated by methyl-binding domain polypeptides immobilized on a magnetic solid matrix. We show that COMPARE-MS could detect five genome equivalents of methylated CGIs in a 1000- to 10,000-fold excess of unmethylated DNA. COMPARE-MS was used to rapidly quantitate hypermethylation at multiple CGIs in >155 prostate tissues, including benign and malignant prostate specimens, and prostate cell lines. This analysis showed that GSTP1, MDR1 and PTGS2 CGI hypermethylation as determined by COMPARE-MS could differentiate between malignant and benign prostate with sensitivities >95% and specificities approaching 100%. This novel technology could significantly improve our ability to detect CGI hypermethylation.
Yegnasubramanian
Srinivasan
S
Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins University School of Medicine 1650 Orleans Street, CRB 116, Baltimore, MD 21231, USA.
Lin
Xiaohui
X
Haffner
Michael C
MC
DeMarzo
Angelo M
AM
Nelson
William G
WG
D000818:Animals; D002460:Cell Line; D045744:Cell Line, Tumor; D011232:Chemical Precipitation; D018899:CpG Islands; D004247:DNA / Q000737:chemistry; D019175:DNA Methylation; D004268:DNA-Binding Proteins / Q000737:chemistry; D019003:Deoxyribonuclease HpaII; D005454:Fluorescence Polarization; D051549:Glutathione S-Transferase pi / Q000235:genetics; D006801:Humans; D008297:Male; D025202:Molecular Diagnostic Techniques; D016133:Polymerase Chain Reaction / Q000379:methods*; D011467:Prostate / Q000378:metabolism; D011471:Prostatic Neoplasms / Q000175:diagnosis*; D017434:Protein Structure, Tertiary; D012372:ROC Curve; D018411:Spodoptera / Q000166:cytology; D013997:Time Factors
D004268:DNA-Binding Proteins; C489325:MBD2 protein, human; D004247:DNA; C496556:GSTP1 protein, human; D051549:Glutathione S-Transferase pi; D019003:Deoxyribonuclease HpaII