論文 - 詳細
| RRC ID | 33390 |
|---|---|
| 著者 | Hashimoto-Gotoh T, Mizuno T, Ogasahara Y, Nakagawa M. |
| タイトル | An oligodeoxyribonucleotide-directed dual amber method for site-directed mutagenesis. |
| ジャーナル | Gene |
| Abstract |
A simple procedure for in vitro site-directed mutagenesis (SDM) was developed using two oligodeoxyribonucleotide (oligo) primers, one serving as selection primer and the other for mutagenesis in the target DNA (mutagenic primer). The mutant clones can be selected by reversion mutations of dual amber (am) to dual Gln codons in the cat gene, resulting in a chloramphenicol-resistant phenotype in sup(O) host cells. Unlike previously reported procedures, the new method requires neither special chemical reagents, nor single-stranded DNA purification, nor any additional biochemical treatments such as multiple PCRs, restriction digestion and so on, but utilises one of the oligo-directed dual am (ODA) plasmids, i.e., pKF16c, pKF17c, pKF18c or pKF19c. The two amber (am) mutations located two codons apart from each other in cat (catam2) can be simultaneously reverted with a 20-nucleotide primer (CQ, pAACCAGACCGTTCAGCTGGA) during primer extension. In a model experiment using the lacZ' gene, an am mutation or a single-bp deletion (sbd) mutation was frequently co-introduced in lacZ' using a mixture of am- or sbd-encoding mutagenic primer for lacZ', and the CQ primer for catam2. Applying this procedure to the human AT cDNA (encoding antithrombin), a missense mutation (Arg406-->Met) in one of the human AT variants, AT-Kyoto, involved in congenital thrombosis disease, was introduced efficiently into the wild-type cDNA (> 85%). |
| 巻・号 | 152(2) |
| ページ | 271-5 |
| 公開日 | 1995-1-23 |
| DOI | 10.1016/0378-1119(94)00750-m |
| PII | 037811199400750M |
| PMID | 7835714 |
| MeSH | Base Sequence DNA, Complementary Genetic Techniques Humans Lac Operon Molecular Sequence Data Mutagenesis, Site-Directed* Plasmids Thrombosis / congenital Thrombosis / genetics |
| IF | 2.984 |
| 引用数 | 78 |
| WOS 分野 | GENETICS & HEREDITY |
| オルトメトリクス指標 |
オルトメトリクス指標項目
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| 最多言及媒体 | Patent(IFI CLAIMS) |
| 各媒体での言及数の合計 | 270 |
| 過去6か月間でのオルトメトリクス指標の変動値 | 6.0 |
| リソース情報 | |
| 原核生物(大腸菌) | pKF16c pKF17c pKF18c pKF19c |