A novel method for sensitive and specific detection of DNA methylation biomarkers based on DNA restriction during PCR cycling

作者：Kneip Christoph; Schmidt Bernd; Fleischhacker Michael; Seegebarth Anke; Lewin Joern; Flemming Nadja; Seemann Stefanie; Schlegel Thomas; Witt Christian; Liebenberg Volker; Dietrich Dimo^*

来源：Biotechniques, 2009, 47(3): 737-741.

DOI：10.2144/000113208

• DNA methylation
• quantitative real-time PCR
• sensitive detection
• diagnostics
• cancer
• early detection
• Tsp509I

DNA methylation is an important epigenetic mechanism involved in fundamental biological processes such as development, imprinting, and carcinogenesis. For these reasons, DNA methylation represents a valuable source for cancer biomarkers. Methods for the sensitive and specific detection of methylated DNA are a prerequisite for the implementation of DNA biomarkers into clinical routine when early detection based on the analysis of body fluids is desired. Here, a novel technique is presented for the detection of DNA methylation biomarkers, based on real-time PCR of bisulfite-treated template with enzymatic digestion of background DNA during amplification using the heat-stable enzyme Tsp509I. An assay for the lung cancer methylation biomarker BARHL2 was used to show clinical and analytical performance of the method in comparison with methylation-specific PCR technology. Both technologies showed comparable performance when analyzing technical DNA mixtures and bronchial lavage samples from 75 patients suspected of having lung cancer. The results demonstrate that the approach is useful for sensitive and specific detection of a few copies of methylated DNA in samples with a high background of unmethylated DNA, such as in clinical samples from body fluids.

• 出版日期2009-9