Abstract

The aim of the study was to develop a sensitive method to identify and quantify methylated DNA fragment in cancer. The genomic DNA of three different tissues (primary cancer of the liver (T-Lv), lung (T-Lg) and kidney (T-Kd)) was analyzed and compared to adjacent normal tissues from the same individuals. After DNA digestion with RsaI, then with a methylation-sensitive and non-sensitive restriction enzymes (HpaII and MspI), digested products were submitted to the AP-PCR amplification. Amplicons were subsequently separated on a Labchip of the Agilent bioanalyzer and the methylated fragments were sized and quantified. We have identified a set of methylated DNA fragments. Although results shown tissue specific variation of methylation level some methylated fragments were common in cancer tissues. There was 100% methylation for a 333 bp fragment in T-Lg and T-Kd and in normal tissues. This same fragment was hypomethylated in T-Lv. Partial hypermethylation of a 357 pb fragment was observed in T-Kd, T-Lv and normal tissues. In T-Lg, methylation was 100 %. A 398 bp fragment was 100 % methylated in T-Kd, but not methylated in T-Lv and T-Lg. The fragment was hypomethylated in normal kidney and liver tissue, 62 % methylated in normal lung tissue. The method is a helpful tool to identify and quantify specific DNA fragments sensitive to methylation.

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