SOLID-PHASE DNA SEQUENCING USING THE BIOTIN AVIDIN SYSTEM
SOLID-PHASE DNA SEQUENCING USING THE BIOTIN AVIDIN SYSTEM
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DOI：
10.1093/nar/16.7.3025
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发表时间：
1988-04-11
影响因子：
14.9
通讯作者：
UHLEN, M
中科院分区：
文献类型：
--
作者：
STAHL, S;HULTMAN, T;UHLEN, M
描述了一种新的固相DNA测序方法。设计了一种定向固定化双链亲和素琼脂糖质粒载体pRIT27。该策略包括在酶作用下将11-bio-dUTP掺入到质粒中，并使用碱进行链特异性洗脱。以固定化的单链DNA为模板进行测序，得到的标记寡核苷酸用碱洗脱。含有固定化模板的亲和凝胶连续用于四种不同的双脱氧-核苷酸反应。固相技术既可用于引物特异性标记，也可用于延伸特异性标记。讨论了该系统在DNA自动测序中应用的可能性。
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.