Abstract

Aberrant DNA methylation of tumor suppressor genes is a frequent epigenetic event that occurs early in tumor progression. Real-time quantitative methylation-specific polymerase chain reaction (QMSP) assays can provide accurate detection and quantitation of methylated alleles that may be potentially useful in diagnosis and risk assessment for cancer. Development of QMSP requires optimization to maximize analytical specificity and sensitivity for the detection of methylated alleles. However, in some cases challenges encountered in primer and probe design can make optimization difficult and limit assay performance. Locked nucleic acids (LNAs) demonstrate increased affinity and specificity for their cognate DNA sequences. In this proof-of-principle study, LNA residues were incorporated into primer and probe design to determine whether LNA-modified oligonucleotides could enhance the analytical performance of QMSP for IGSF4 promoter methylation in human cancer cell lines using either SYBR Green or fluorogenic probe detection methods. Use of LNA primers in QMSP with SYBR Green improved analytical specificity for methylated alleles and eliminated the formation of nonspecific products because of mispriming from unmethylated alleles. QMSP using LNA probe and primers showed an increased amplification efficiency and maximum fluorescent signal. QMSP with LNA oligonucleotides and either detection method could reliably detect five genome equivalents of methylated DNA in 1000- to 10,000-fold excess unmethylated DNA. Thus, LNA oligonucleotides can be used in QMSP optimization to enhance analytical performance.