A novel technique for the identification of CpG islands exhibiting altered methylation patterns (ICEAMP).
Aberrant CpG methylation changes occurring during tumour progression include the loss (hypomethylation) and gain (hypermethylation) of methyl groups. Techniques currently available for examining such changes either require selection of a region, then examination of methylation changes, or utilise methylation-sensitive restriction enzymes to identify an alteration. We describe here a novel method that identifies genomic regions as a consequence of altered methylation during tumourigenesis. A methyl-CpG binding domain column isolates methylated GC-rich sequences from both tumours and surrounding normal tissue. Subsequent subtractive hybridisation removes sequences common to both, leaving only methylated sequences unique to the tumour. Libraries of sequences generated using DNA derived from a breast tumour (histological grade; poorly differentiated) as 'tester' and from matched normal tissue as 'driver' were examined; 26% of clones had the sequence criteria of a CpG island (CGI). Analysis using the bisulfite technique revealed that a number of these sequences were methylated in tumour DNA relative to the normal control. We have therefore demonstrated the ability of this technique, the identification of CGI exhibiting altered methylation patterns (ICEAMP), to isolate tumour-specific methylated GC-rich sequences. This will allow a comprehensive identification of methylation changes during tumourigenesis and will lead to a better understanding of the processes involved.
Brock
G J
GJ
Division of Molecular Genetics, Institute of Biomedical and Life Sciences, University of Glasgow, 56 Dumbarton Road, Glasgow G11 6NU, UK. gbrock@molgen.gla.ac.uk
Huang
T H
TH
Chen
C M
CM
Johnson
K J
KJ
D001482:Base Composition; D001943:Breast Neoplasms / Q000235:genetics; D002853:Chromatography, Liquid / Q000379:methods*; D018899:CpG Islands / Q000235:genetics*; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019175:DNA Methylation; D005260:Female; D006801:Humans; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization / Q000379:methods; D016133:Polymerase Chain Reaction / Q000379:methods; D017422:Sequence Analysis, DNA
D004247:DNA