論文 - 詳細
| RRC ID | 7154 |
|---|---|
| 著者 | Liu XP, Liu JH. |
| タイトル | The terminal 5' phosphate and proximate phosphorothioate promote ligation-independent cloning. |
| ジャーナル | Protein Sci |
| Abstract |
Function studies of many proteins are waited to develop after genome sequencing. High-throughout technology of gene cloning will strongly promote proteins' function studies. Here we describe a ligation-independent cloning (LIC) method, which is based on the amplification of target gene and linear vector by PCR using phosphorothioate-modified primers and the digestion of PCR products by lambda exonuclease. The phosphorothioate inhibits the digestion and results in the generation of 3' overhangs, which are designed to form complementary double-stranded DNA between target gene and linear vector. We compared our phosphorothioate primer cloning methods with several LIC methods, including dU primer cloning, hybridization cloning, T4 DNA polymerase cloning, and in vivo recombination cloning. The cloning efficiency of these LIC methods are as follows: phosphorothioate primer cloning > dU primer cloning > hybridization cloning > T4 DNA polymerase cloning >> in vivo recombination cloning. Our result shows that the 3' overhangs is a better cohesive end for LIC than 5' overhang and the existence of 5'phosphate promotes DNA repair in Escherichia coli, resulting in the improvement of cloning efficiency of LIC. We succeeded in constructing 156 expression plasmids of Aeropyrum pernix genes within a week using our method. |
| 巻・号 | 19(5) |
| ページ | 967-73 |
| 公開日 | 2010-5-1 |
| DOI | 10.1002/pro.374 |
| PMID | 20217896 |
| PMC | PMC2868239 |
| MeSH | Aeropyrum / genetics* Aeropyrum / metabolism Cloning, Molecular / methods* Exonucleases / metabolism Genes, Bacterial Phosphorothioate Oligonucleotides / chemistry Phosphorothioate Oligonucleotides / metabolism* |
| IF | 3.876 |
| 引用数 | 15 |
| WOS 分野 | BIOCHEMISTRY & MOLECULAR BIOLOGY |
| 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 | 4 |
| 過去6か月間でのオルトメトリクス指標の変動値 | 0.0 |
| リソース情報 | |
| 一般微生物 | JCM 9820 |