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Preparation of Phi29 DNA Polymerase Free of Amplifiable DNA Using Ethidium Monoazide, an Ultraviolet-Free Light-Emitting Diode Lamp and Trehalose
https://repo.qst.go.jp/records/46727
https://repo.qst.go.jp/records/46727580ab276-e5e3-4f3a-b958-f44bf763429b
| Item type | 学術雑誌論文 / Journal Article(1) | |||||
|---|---|---|---|---|---|---|
| 公開日 | 2014-02-19 | |||||
| タイトル | ||||||
| タイトル | Preparation of Phi29 DNA Polymerase Free of Amplifiable DNA Using Ethidium Monoazide, an Ultraviolet-Free Light-Emitting Diode Lamp and Trehalose | |||||
| 言語 | ||||||
| 言語 | eng | |||||
| 資源タイプ | ||||||
| 資源タイプ識別子 | http://purl.org/coar/resource_type/c_6501 | |||||
| 資源タイプ | journal article | |||||
| アクセス権 | ||||||
| アクセス権 | metadata only access | |||||
| アクセス権URI | http://purl.org/coar/access_right/c_14cb | |||||
| 著者 |
Takahashi, Hirokazu
× Takahashi, Hirokazu× Yamazaki, Hiroyuki× Akanuma, Satoshi× Kanahara, Hiroko× Saito, Toshiyuki× Chimuro, Tomoyuki× Kobayashi, Takayoshi× Ohtani, Toshio× Yamamoto, Kimiko× Sugiyama, Shigeru× 齋藤 俊行 |
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| 抄録 | ||||||
| 内容記述タイプ | Abstract | |||||
| 内容記述 | 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. | |||||
| 書誌情報 |
PLOS ONE 巻 9, 号 2, p. e82624, 発行日 2014-02 |
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| 出版者 | ||||||
| 出版者 | PLOS | |||||
| ISSN | ||||||
| 収録物識別子タイプ | ISSN | |||||
| 収録物識別子 | 1932-6203 | |||||
| DOI | ||||||
| 識別子タイプ | DOI | |||||
| 関連識別子 | 10.1371/journal.pone.0082624 | |||||
| 関連サイト | ||||||
| 識別子タイプ | URI | |||||
| 関連識別子 | http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0082624 | |||||
| 関連名称 | http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0082624 | |||||
