Methylated-CpG island recovery assay: a new technique for the rapid detection of methylated-CpG islands in cancer

Lab Invest. 2005 Sep;85(9):1172-80. doi: 10.1038/labinvest.3700311.

Abstract

Hypermethylation of CpG islands is a phenomenon commonly observed during the development and progression of human tumors. Detection of methylated-CpG islands in easily accessible biological materials such as serum has the potential to be useful for the early diagnosis of cancer. Most currently used methods for detecting methylated-CpG islands are based on sodium bisulfite conversion of genomic DNA, followed by PCR reactions. Here we describe a method, methylated-CpG island recovery assay (MIRA) that does not depend on the use of sodium bisulfite but has similar sensitivity and specificity as bisulfite-based approaches. Methyl-CpG-binding domain proteins, such as methyl-CpG-binding domain protein-2 (MBD2), have the capacity to bind specifically to methylated DNA sequences. In the MIRA procedure, sonicated genomic DNA isolated from cells or tissue is incubated with a matrix containing glutathione-S-transferase-MBD2b in the presence of methyl-CpG-binding domain protein 3-like-1, a binding partner of MBD2 that increases the affinity of MBD2 for methylated DNA. Specifically bound DNA is eluted from the matrix and gene-specific PCR reactions are performed to detect CpG island methylation. Methylation can be detected using 1 ng of DNA or 3000 cells. MIRA is a specific and sensitive, but not laborious, technique that can be clinically useful in the detection and diagnosis of any DNA methylation-associated disease, including cancer.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Base Sequence
  • CpG Islands*
  • DNA / genetics
  • DNA Methylation*
  • Humans
  • Molecular Sequence Data
  • Neoplasms / genetics*
  • Plasmids
  • Polymerase Chain Reaction
  • Promoter Regions, Genetic
  • Sensitivity and Specificity

Substances

  • DNA