Solid phase DNA sequencing using the biotin-avidin system.
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.
Ståhl
S
S
Department of Biochemistry, Royal Institute of Technology, Stockholm, Sweden.
Hultman
T
T
Olsson
A
A
Moks
T
T
Uhlén
M
M
D001322:Autoanalysis / Q000379:methods; D001360:Avidin; D001483:Base Sequence; D001710:Biotin; D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000302:isolation & purification*; D003854:Deoxyribonucleotides; D004800:Enzymes, Immobilized; D005822:Genetic Vectors; D010957:Plasmids; D012685:Sepharose; D012972:Sodium Hydroxide
D003854:Deoxyribonucleotides; D004800:Enzymes, Immobilized; D001360:Avidin; D012972:Sodium Hydroxide; D001710:Biotin; D004247:DNA; D012685:Sepharose