Solid phase DNA sequencing using the biotin-avidin system

• Published in: Nucleic acids research Vol. 16; no. 7; pp. 3025 - 3038
• Main Authors: STAHL, S, HULTMAN, T, OLSSON, A, MOKS, T, UHLEN, M
• Format: Journal Article
• Language: English
• Published: Oxford Oxford University Press 11-04-1988
• Subjects: Analytical, structural and metabolic biochemistry; Autoanalysis - methods; Avidin; Base Sequence; Biological and medical sciences; Biotin; Cloning, Molecular - methods; Deoxyribonucleotides; DNA; DNA - isolation & purification; Dna, deoxyribonucleoproteins; Enzymes, Immobilized; Fundamental and applied biological sciences. Psychology; Genetic Vectors; Nucleic acids; nucleotide sequence; Plasmids; Sepharose; Sodium Hydroxide; Primary structure; Solid phase; Investigation method; DNA
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/16.7.3025

A novel method for solid-phase DNA sequencing is described. A plasmid vector, pRIT27, has been designed to allow directional immobilization of double stranded plasmid to avidin agarose. The strategy involves enzymatic incorporation of 11-bio-dUTP into the plasmid and strand specific elution using alkali. The immobilized single stranded DNA is used as template for sequencing reactions and the resulting labelled oligonucleotides are eluted by alkali. The affinity gel containing the immobilized template is consecutively used for the four different dideoxy-nucleotide reactions. The solid-phase technique can be used for both primer specific or extension specific labelling. The possibility to use the system in automated DNA sequencing is discussed.