Abstract
BackgroundDNA methylation changes are widely used as early molecular markers in cancer detection. Sensitive detection and classification of rare methylation changes in DNA extracted from circulating body fluids or complex tissue samples is crucial for the understanding of tumor etiology, clinical diagnosis and treatment. In this paper, we describe a combined method to monitor the presence of methylated tumor DNA in an excess of unmethylated background DNA of non-tumorous cells. The method combines heavy methyl-PCR, which favors preferential amplification of methylated marker sequence from bisulfite-treated DNA with a methylation-specific single nucleotide primer extension monitored by ion-pair, reversed-phase, high-performance liquid chromatography separation.ResultsThis combined method allows detection of 14 pg (that is, four to five genomic copies) of methylated chromosomal DNA in a 2000-fold excess (that is, 50 ng) of unmethylated chromosomal background, with an analytical sensitivity of > 90%. We outline a detailed protocol for the combined assay on two examples of known cancer markers (SEPT9 and TMEFF2) and discuss general aspects of assay design and data interpretation. Finally, we provide an application example for rapid testing on tumor methylation in plasma DNA derived from a small cohort of patients with colorectal cancer.ConclusionThe method allows unambiguous detection of rare DNA methylation, for example in body fluid or DNA isolates from cells or tissues, with very high sensitivity and accuracy. The application combines standard technologies and can easily be adapted to any target region of interest. It does not require costly reagents and can be used for routine screening of many samples.
Highlights
DNA methylation changes are widely used as early molecular markers in cancer detection
We present a novel assay combining sensitive HMPCR with methylation-restricted single nucleotide primer extension (MR-SNuPE) followed by ion-pair, reversed-phase, high-performance liquid chromatography IIP/RP-HPLC) detection
We first determined the limits of SNuPE detection by IP/ RP-HPLC on PCR products obtained by conventional PCR of bisulfite-treated template DNA
Summary
DNA methylation changes are widely used as early molecular markers in cancer detection. Changes in DNA methylation such as hypermethylation of tumor suppressor genes are regarded as early molecular events during cancer development. Such epigenetic changes are widely used as molecular markers in tumor cell diagnostics [1,2,3]. Selective and robust PCR-based screening methods for very early detection of abnormal tumor-specific methylation become increasingly important. Methods to screen for the low abundance of aberrantly methylated tumor DNA present in peripheral blood samples or other body fluids are regarded as very promising, non-invasive, early cancer detection tests [4]. HPLC (Target detection by peak analysis) routine diagnostics in a pilot study on two pre-validated colon cancer methylation markers, transmembrane epidermal growth factor (TMEFF2/TPEF) and septin 9 (SEPT9) [14,15,16,17,18]
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