Optimized conditions for solid-phase sequencing: simultaneous chemical cleavage of a series of long DNA fragments immobilized on CCS anion-exchange paper |
| |
| Authors: | A Rosenthal R Jung H D Hunger |
| |
| Affiliation: | 1. Dalian Key Laboratory of Genetic Resources for Marine Shellfish, Liaoning Ocean and Fisheries Science Research Institute, Dalian, China;2. Key Laboratory of Protection and Utilization of Aquatic Germplasm Resource, Ministry of Agriculture and Rural Affairs, Dalian, China;3. Key Laboratory of Plant Biotechnology of Liaoning Province, School of Life Sciences, Liaoning Normal University, Dalian, China;4. Dalian Changhai-Yide Aquatic Products Co., LTD, Dalian, China;5. Institute of Marine and Environmental Technology, Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, USA;6. Key Laboratory of Mariculture, Ministry of Education, Ocean University of China, Qingdao, China;7. College of Fisheries and Life Science, Dalian Ocean University, Dalian, China |
| |
| Abstract: | A solid-phase method for simultaneous sequencing of ten or more long DNA fragments has been developed, using as support the cellulose matrix for chemical sequencing (CCS), anion-exchange paper [Rosenthal et al., Nucl. Acids Res. 13 (1985) 1173-1184]. We optimized several of the seven steps which include: (i) immobilization; (ii) washing; (iii) modification; (iv) washing; (v) sorting of the paper segments; (vi) piperidine reaction and chemical elution, and (vii) lyophilization. During carrier-supported chemical cleavage with dimethylsulfate (DMS) (G), HCOOH (A + G), KMnO4 (T greater than Pu) and NH2OH (C), losses of immobilized DNA are very low. DNA fragments ranging in length from several hundred bp up to 6 kb can be effectively chemically eluted from CCS paper during the piperidine reaction with an efficiency of more than 90%. Because no DNA salt elution and ethanol precipitation steps are necessary the method is rapid, convenient and allows complete automation. |
| |
| Keywords: | |
| 本文献已被 ScienceDirect 等数据库收录！ |
|
