title: Preparation of Phi29 DNA polymerase free of amplifiable DNA using ethidium monoazide, an ultraviolet-free light-emitting diode lamp and trehalose.
abstract: We previously reported that multiply-primed rolling circle amplification (MRPCA) using modified random RNA primers can amplify tiny amounts of circular DNA without producing any byproducts. However, contaminating DNA in recombinant Phi29 DNA polymerase adversely affects the outcome of MPRCA, especially for negative controls such as non-template controls. The amplified DNA in negative control casts doubt on the result of DNA amplification. Since Phi29 DNA polymerase has high affinity for both single-strand and double-stranded DNA, some amount of host DNA will always remain in the recombinant polymerase. Here we describe a procedure for preparing Phi29 DNA polymerase which is essentially free of amplifiable DNA. This procedure is realized by a combination of host DNA removal using appropriate salt concentrations, inactivation of amplifiable DNA using ethidium monoazide, and irradiation with visible light from a light-emitting diode lamp. Any remaining DNA, which likely exists as oligonucleotides captured by the Phi29 DNA polymerase, is degraded by the 3'-5' exonuclease activity of the polymerase itself in the presence of trehalose, used as an anti-aggregation reagent. Phi29 DNA polymerase purified by this procedure has little amplifiable DNA, resulting in reproducible amplification of at least ten copies of plasmid DNA without any byproducts and reducing reaction volume. This procedure could aid the amplification of tiny amounts DNA, thereby providing clear evidence of contamination from laboratory environments, tools and reagents.
authors: Nanobiotechnology Laboratory, Food Engineering Division, National Food Research Institute, National Agriculture and Food Research Organization, Tsukuba, Ibaraki, Japan.
authors: Hirokazu
authors: H
authors: Takahashi
authors: Isehara Research Laboratory, Technology & Development Division, Kanto Chemical Co., Inc., Isehara, Kanagawa, Japan.
authors: Hiroyuki
authors: H
authors: Yamazaki
authors: Nanobiotechnology Laboratory, Food Engineering Division, National Food Research Institute, National Agriculture and Food Research Organization, Tsukuba, Ibaraki, Japan.
authors: Satoshi
authors: S
authors: Akanuma
authors: Nanobiotechnology Laboratory, Food Engineering Division, National Food Research Institute, National Agriculture and Food Research Organization, Tsukuba, Ibaraki, Japan.
authors: Hiroko
authors: H
authors: Kanahara
authors: Transcriptome Profiling Group, National Institute of Radiological Sciences, Chiba, Chiba, Japan.
authors: Toshiyuki
authors: T
authors: Saito
authors: Isehara Research Laboratory, Technology & Development Division, Kanto Chemical Co., Inc., Isehara, Kanagawa, Japan.
authors: Tomoyuki
authors: T
authors: Chimuro
authors: Bio-Chemical Department, Reagent Division, Kanto Chemical Co., Inc. Tokyo, Japan.
authors: Takayoshi
authors: T
authors: Kobayashi
authors: Nanobiotechnology Laboratory, Food Engineering Division, National Food Research Institute, National Agriculture and Food Research Organization, Tsukuba, Ibaraki, Japan.
authors: Toshio
authors: T
authors: Ohtani
authors: Insect Genome Laboratory, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, Japan.
authors: Kimiko
authors: K
authors: Yamamoto
authors: Nanobiotechnology Laboratory, Food Engineering Division, National Food Research Institute, National Agriculture and Food Research Organization, Tsukuba, Ibaraki, Japan.
authors: Shigeru
authors: S
authors: Sugiyama
authors: Nanobiotechnology Laboratory, Food Engineering Division, National Food Research Institute, National Agriculture and Food Research Organization, Tsukuba, Ibaraki, Japan.
authors: Toshiro
authors: T
authors: Kobori
mesh_terms: D001386:Azides / Q000737:chemistry*; D001435:Bacteriophages / Q000201:enzymology* / Q000235:genetics; D004279:DNA, Viral / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D008027:Light; D014199:Trehalose; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification
chemical_list: D001386:Azides; D004279:DNA, Viral; D014764:Viral Proteins; C014535:8-azidoethidium; D014199:Trehalose; D004259:DNA-Directed DNA Polymerase