title: Treatment of PCR products with exonuclease I and heat-labile alkaline phosphatase improves the visibility of combined bisulfite restriction analysis.
abstract: DNA methylation plays a vital role in the regulation of gene expression. Abnormal promoter hypermethylation is an important mechanism of inactivating tumor suppressor genes in human cancers. Combined bisulfite restriction analysis (COBRA) is a widely used method for identifying the DNA methylation of specific CpG sites. Here, we report that exonuclease I and heat-labile alkaline phosphatase can be used for PCR purification for COBRA, improving the visibility of gel electrophoresis after restriction digestion. This improvement is observed when restriction digestion is performed at a high temperature, such as 60 degrees C or 65 degrees C, with BstUI and TaqI, respectively. This simple method can be applied instead of DNA purification using spin columns or phenol/chloroform extraction. It can also be applied to other situations when PCR products are digested by thermophile-derived restriction enzymes, such as PCR restriction fragment length polymorphism (RFLP) analysis.
authors.affiliation: Department of Respiratory Medicine, The University of Tokyo Hospital, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-8655, Japan.
authors.forename: Kousuke
authors.initials: K
authors.lastname: Watanabe
authors.forename: Noriko
authors.initials: N
authors.lastname: Emoto
authors.forename: Mitsuhiro
authors.initials: M
authors.lastname: Sunohara
authors.forename: Masanori
authors.initials: M
authors.lastname: Kawakami
authors.forename: Hidenori
authors.initials: H
authors.lastname: Kage
authors.forename: Takahide
authors.initials: T
authors.lastname: Nagase
authors.forename: Nobuya
authors.initials: N
authors.lastname: Ohishi
authors.forename: Daiya
authors.initials: D
authors.lastname: Takai
mesh_terms: D000469:Alkaline Phosphatase / Q000737:chemistry*; D000818:Animals; D002460:Cell Line; D018899:CpG Islands; D004247:DNA / Q000032:analysis* / Q000737:chemistry; D019175:DNA Methylation; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D005090:Exodeoxyribonucleases / Q000737:chemistry*; D006358:Hot Temperature; D006801:Humans; D016133:Polymerase Chain Reaction; D013447:Sulfites / Q000737:chemistry
chemical_list: D013447:Sulfites; D004247:DNA; D005090:Exodeoxyribonucleases; C028450:exodeoxyribonuclease I; C039566:CGCG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific; C027046:TCGA-specific type II deoxyribonucleases; D000469:Alkaline Phosphatase; C042345:hydrogen sulfite