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New quantitative Methylation‐Sensitive Arbitrarily Primed PCR (qMS AP‐PCR) method to determine DNA methylation level in cancer
Author(s) -
Bousserouel Souad,
Chango Abalo
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.4.a151-b
Subject(s) - microbiology and biotechnology , hpaii , methylation , dna methylation , dna , genomic dna , amplicon , restriction enzyme , biology , chemistry , polymerase chain reaction , biochemistry , gene , gene expression
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.