Fingerprinting Technique Carcinomas Using the Methylation-sensitive Restriction Identification of DNA Methylation Markers for Human Breast

igenesis, abnormally hypermethylated or hypomethylated sequences occurring in the tumor genome need to be identified. In this study, we have developed a PCR-based method, called MSRF,3 to identify abnormally methylated CpG sites in breast carci nomas. Initially, genomic DNA was digested with a 4-base restriction endonuclease known to cut bulk DNA into small fragments but rarely to cut in the CG-rich regions. The digests were treated with a second restriction endonuclease, which discriminates between methylated and unmethylated CpG sites, and then amplified by PCR with short arbitrary primers (lO-mers) at low stringency. Amplified products or DNA fingerprints were resolved by high-resolution gel electrophore sis, and aberrantly methylated patterns were detected in the amplified tumor DNA relative to the amplified normal DNA of the same patient. Here we have successfully employed the technique to identify and clone genomic fragments that frequently undergo methylation changes in primary breast carcinomas. Materials and Methods Tissue Samples. Tumor specimens were obtained from patients with in vasive breast carcinomas undergoing mastectomies or biopsies at the Ellis Fischel Cancer Center (Columbia, MO; this study has been approved by our institutional review board). The adjacent nonneoplastic breast tissue was also obtained from the same patient to serve as a control (designated as â€oenormal†•). High molecular weight DNA was extracted using standard methods. MSRF. Genomic DNA was digested with MseI alone or digested with BstU I and MseI at 10 units per @tgDNA following the conditions recom mended by the supplier (New England Biolabs). The PCR reaction was performed with the digested DNA (20†" 100ng) in a 20-s.d volume containing a pair of primers (0.4 jIM), 0.15 units of Taq DNA polymerase (Life Tech nologies, Inc.), 5% (v/v) DMSO, 200 p.M deoxynucleotide triphosphates, and 2 @Ci [a-32P] dCTP (3000 Ci/mmol; Amersham) in buffer III [30 mM Tricine (pH 8.4), 2 mM MgCl2, 5 mr@i f3-mercaptoethanol,0.01% (w/v) gelatin, and 0.1% (w/v) thesiti (13). Initial denaturation was for 5 mm at 94°C.DNA samples were then subjected to 30 cycles of amplification consisting of 2 mm of denaturation at 94°C, 1 mm of annealing at 40°C,and 2 mm of extension at 72°Cin a PTC-l00 thermo cycler (M. J. Research, Inc.). The final extension was lengthened to 10 mm. The products (20 pA)of each amplification reaction were mixed with 5 pi of loading dye. Four pAof each DNA sample were size fractionated on a 4.5% nondenaturing polyacrylamide gel. Samples were loaded …