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Changes in methylation patterns identified by two‐dimensional DNA fingerprinting
Author(s) -
Uhlmann Karen,
Marczinek Karola,
Hampe Jochen,
Thiel Gundula,
Nürnberg Peter
Publication year - 1999
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/(sici)1522-2683(19990101)20:8<1748::aid-elps1748>3.0.co;2-1
Subject(s) - dna methylation , dna profiling , dna , genetics , biology , methylated dna immunoprecipitation , methylation , computational biology , gene , gene expression
Two‐dimensional DNA fingerprinting (2‐D fingerprinting) is a sensitive tool for genomic difference analysis between tumor DNA and constitutive DNA of glioma patients. Numerous differences were found even in low‐grade gliomas. They can be interpreted as deletions, amplifications, rearrangements, Hae III restriction site mutations, tandem repeat instabilities, or methylation differences. The influence of methyl groups on the melting behavior of double‐stranded DNA fragments in a denaturing gradient gel was demonstrated by analyzing the migration of λ‐phage DNA fragments in 2‐D fingerprint gels. A characteristic intensity shift between two neighboring spots in several glioma samples was identified and verified by rehybridization of 2‐D filters with a cloned DNA fragment corresponding to the lower spot in 10 out of 11 pilocytic astrocytomas. We hypothesized that this shift may be related to an alteration in the methylation pattern of the tumor DNA. This was specifically tested by analyzing the underlying 750 bp genomic fragment (including 21 CpG dinucleotides) with bisulfite treatment of agarose‐embedded DNA. A methylation grade of 88% in tumor DNA as compared to 96% in blood DNA was found. Although only one CpG is located in the melting domain of the cloned fragment, this particular CpG is methylated in all blood samples, but mostly demethylated in the tumor samples. In conclusion, we demonstrate that 2‐D fingerprinting may be a powerful tool for the detection of DNA methylation changes in genomic difference analysis.