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Optimized conditions for solid-phase sequencing: simultaneous chemical cleavage of a series of long DNA fragments immobilized on CCS anion-exchange paper.

Authors
  • Rosenthal, A
  • Jung, R
  • Hunger, H D
Type
Published Article
Journal
Gene
Publisher
Elsevier
Publication Date
Jan 01, 1986
Volume
42
Issue
1
Pages
1–9
Identifiers
PMID: 3721198
Source
Medline
License
Unknown

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.

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