Accelerated bisulfite-deamination of cytosine in the genomic sequencing procedure for DNA methylation analysis.
Understanding the biological consequences of DNA methylation is a current focus of intensive studies. A standard method for analyzing the methylation at position 5 of cytosines in genomic DNA involves chemical modification of the DNA with bisulfite, followed by PCR amplification and sequencing. Bisulfite deaminates cytosine, but it deaminates 5-methylcytosine only very slowly, thereby allowing determination of the methylated sites. The determination is usually performed using sodium bisulfite solutions of 3-5 M concentration with an incubation period of 12-16 hr at 50 degrees C. We demonstrate here that this deamination can be speeded up significantly by increasing the bisulfite concentration and the temperature with which the reaction is performed. In an experiment, in which denatured DNA was treated with 9 M bisulfite for 10 min at pH 5.4 and 90 degrees C, deamination of cytosines occurred to an extent of 99.6%, while 5-methylcytosine residues in the DNA were deaminated at less than 10%. Using a plasmid DNA fragment, we observed that the DNA can serve as a template for PCR amplification after the bisulfite treatment. This new procedure is expected to offer an improved genomic sequencing method, leading to the promotion of research on understanding the biological and medical significance of DNA methylation.
Hayatsu
Hikoya
H
Shujitsu University, School of Pharmacy, 1-6-1 Nishigawara, Okayama 703-8516, Japan.
Negishi
Kazuo
K
Shiraishi
Masahiko
M
D000818:Animals; D001482:Base Composition; D002851:Chromatography, High Pressure Liquid; D003596:Cytosine / Q000378:metabolism*; D019175:DNA Methylation; D003641:Deamination; D016678:Genome; D008297:Male; D009705:Nucleosides / Q000032:analysis; D012474:Salmon; D017422:Sequence Analysis, DNA; D013447:Sulfites / Q000378:metabolism*; D013737:Testis
D009705:Nucleosides; D013447:Sulfites; D003596:Cytosine; C042345:hydrogen sulfite