C T

Fig. 2. Schematic diagram representing steps involved in bisulfite genomic sequencing.

and carcinogenesis.8 Aberrations in DNA methylation pattern characterized by global hypomethylation and regional hypermethylation occur in many human malignancies. In normal cells CpG islands in the promoter regions of most of the tumor suppressor genes, for example, those encoding pi6, pl5, retinoblastoma protein (Rb), and von Hippel-Lindau protein (VHL) are unmethylated and expressed. In cancer cells one or more of these tumor suppressor genes are rendered nonfunctional due to point mutations in their coding region. In contrast, some of the tumor suppressor genes in many human tumors are silenced by methylation of CpG islands in their promoters.9 The signal that initiates alteration in methylation profile is yet to be understood.

A method exists to identify the methylated cytosines at specific sites within a gene. Bisulfite genomic sequencing, originally developed by Clark et al.,w is an innovative technique that can be used effectively to determine the methylation

9 J.-P. Issa, Curr. Topics Microbiol. Immunol. 249, 101 (1999).

10 S. J. Clark, J. Harrison, C. L. Paul, and M. Frommer, Nucleic Acids Res. 22, 2990 (1994).

status of specific CpG base pairs in a specific gene. Briefly, the procedure consists of denaturation, sulfonation, desulfonation, and polymerase chain reaction (PCR) amplification followed by restriction enzyme digestion and sequencing (see Fig. 2). Treatment of genomic DNA with bisulfite reagent under denaturing conditions converts cytosines in the DNA to uracils, amplified as thymine during subsequent PCR. Therefore, the few remaining cytosines in the sequencing gel are due to either 5-methylcytosine in the chromosomal DNA or incomplete bisulfite conversion, a common problem in this reaction. To overcome this artifact we have modified the original protocol. Denaturing DNA at 95° for 2 min after 30 min of desulfonation during this procedure facilitates completion of the reaction. Next, the amplified DNA is digested with several restriction enzymes before sequencing to check complete bisulfite conversion.

Methodology

Preparation of Genomic DNA from Cells or Tissues

Chromosomal DNA is isolated from mouse lymphosarcoma cells by lysis in digestion buffer [100 mM NaCl, 10 mM Tris-HCl (pH 8), 25 mM EDTA (pH 8), 0.5% (w/v) sodium dodecyl sulfate (SDS)] followed by overnight digestion with proteinase K (0.2 mg/ml) at 37°, phenol-chloroform extraction, and ethanol precipitation. The isolated DNA is allowed to dissolve overnight in TE [10 mMTris-HCl (pH 8), 1 mM EDTA (pH 8)] buffer. To isolate DNA from tissues, pulverized frozen tissues are homogenized in the DNA digestion buffer before proteinase K digestion. Enough tissue or cells should be used to obtain at least 5-10 /xg of DNA. For a detailed method for DNA isolation see Ausubel et al.u

Conversion of Unmethylated Cytosines of Genomic DNA to Uracils by Treatment with Bisulfite Reagent

The protocol for bisulfite genomic sequencing is shown in Fig. 3. Genomic DNA (5 Mg) is denatured with 0.3 M NaOH (freshly prepared) at 37° for 30 min. PCR tubes (0.2 to 0.5 ml) are used for this reaction (see Fig. 3). The denatured DNA is treated with freshly prepared sodium metabisulfite (Sigma, St. Louis, MO) solution (final concentration, 2.35 M) containing hydroquinone (Sigma) (final concentration, 0.04 M) in a thermal cycler 20 times at 95° for 2 min and at 50° for 30 min. Thermal denaturation of DNA every 30 min at 95° prevents formation of double-stranded DNA (dsDNA), which is resistant to sulfonation by bisulfite reagent. This protocol results in complete conversion of cytosines to uracil. The bisulfite-treated DNA is purified with a Wizard DNA clean-up kit (Promega, Madison, WI), eluted in 50 ¿¿1 of water, and treated with 50 mMNaOH (final concentration) in a volume of 100 /xl at 37° for 30 min for desulfonation. DNA is then

11 F. M. Ausubel, R. Brent, R. E. Kingston, and D. D. Moore (eds.), "Current Protocols in Molecular Biology," Section 2.2.1. John Wiley & Sons, New York, 1987.

Genomic DNA (5 (xg in 10 |il) in 0.2 to 0.5 ml tubes i

Add 2 nl of freshly prepared 2 M NaOH

0 0

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