Parallel assessment of CpG methylation by two-color hybridization with oligonucleotide arrays.
We have developed a method for the parallel analysis of multiple CpG sites in genomic DNA for their state of methylation. Hypermethylation of CpG islands within the promoters and 5' exons of genes has been found to be a mechanism of transcriptional inactivation associated with a variety of tumors. The method that we developed relies on the differential reactivity of methylated and unmethylated cytosines with sodium bisulfite, which exclusively converts unmethylated cytosines to deoxyuracils. The resulting sequence changes are determined with single-nucleotide resolution by hybridization to an oligonucleotide array. Cohybridization with a reference sample containing a different label provides an internal standard for assessment of methylation state. This method provides advantages in parallelism over existing methods of methylation analysis. We have demonstrated this technique with a region from the promoter of the tumor suppressor gene p16, which is hypermethylated in many cancers.
Balog
Robert P
RP
Center for Biomedical Inventions, University of Texas Southwestern Medical Center at Dallas, Dallas, TX 75390-8573, USA.
de Souza
Y Emi Ponce
YE
Tang
Hue M
HM
DeMasellis
Gina M
GM
Gao
Boning
B
Avila
Adrian
A
Gaban
Desmond J
DJ
Mittelman
David
D
Minna
John D
JD
Luebke
Kevin J
KJ
Garner
Harold R
HR
D001483:Base Sequence; D002232:Carbocyanines / Q000737:chemistry; D003001:Cloning, Molecular; D018899:CpG Islands; D003596:Cytosine / Q000032:analysis / Q000737:chemistry; D019175:DNA Methylation; D017931:DNA Primers; D004273:DNA, Neoplasm / Q000737:chemistry; D016147:Genes, Tumor Suppressor; D006801:Humans; D009693:Nucleic Acid Hybridization / Q000379:methods; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D011401:Promoter Regions, Genetic; D012015:Reference Standards; D013447:Sulfites / Q000737:chemistry; D014407:Tumor Cells, Cultured
D002232:Carbocyanines; D017931:DNA Primers; D004273:DNA, Neoplasm; D013447:Sulfites; C089187:cyanine dye 3; C085321:cyanine dye 5; D003596:Cytosine; C009279:sodium bisulfite