Highly efficient preparation of single-stranded DNA rings by T4 ligase at abnormally low Mg(II) concentration
| Autor: | Ran An, Yiqiao Fan, Qi Li, Xingguo Liang, Xiaoming Pan, Makoto Komiyama, Jing Li |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
DNA Ligases DNA Single-Stranded Biology In Vitro Techniques 010402 general chemistry Ring (chemistry) 01 natural sciences Polymerization 03 medical and health sciences chemistry.chemical_compound Genetics Magnesium Cloning Molecular Cloning chemistry.chemical_classification DNA ligase Base Sequence Intermolecular force Osmolar Concentration Substrate (chemistry) Combinatorial chemistry 0104 chemical sciences 030104 developmental biology Biochemistry chemistry Cyclization Methods Online DNA Circular Selectivity DNA |
| Zdroj: | Nucleic Acids Research |
| ISSN: | 1362-4962 0305-1048 |
| Popis: | Preparation of large amount of single-stranded circular DNA in high selectivity is crucial for further developments of nanotechnology and other DNA sciences. Herein, a simple but practically useful methodology to prepare DNA rings has been presented. One of the essential factors is to use highly diluted T4 ligase buffer for ligase reactions. This strategy is based on our unexpected finding that, in diluted T4 buffers, intermolecular polymerization of DNA fragments is greatly suppressed with respect to their intramolecular cyclization. This promotion of cyclization is attributable to abnormally low concentration of Mg2+ ion (0.5–1.0 mM) but not ATP in the media for T4 ligase reactions. The second essential factor is to add DNA substrate intermittently to the mixture and maintain its temporal concentration low. By combining these two factors, single-stranded DNA rings of various sizes (31–74 nt) were obtained in high selectivity (89 mol% for 66-nt DNA) and in satisfactorily high productivity (∼0.2 mg/ml). A linear 72-nt DNA was converted to the corresponding DNA ring in nearly 100% selectivity. The superiority of this new method was further substantiated by the fact that small-sized DNA rings (31–42 nt), which were otherwise hardly obtainable, were successfully prepared in reasonable yields. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|