Ståhl S, Hultman T, Olsson A, Moks T, Uhlén M
Department of Biochemistry, Royal Institute of Technology, Stockholm, Sweden.
Nucleic Acids Res. 1988 Apr 11;16(7):3025-38. 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.
本文描述了一种用于固相DNA测序的新方法。已设计出一种质粒载体pRIT27，以实现双链质粒向抗生物素蛋白琼脂糖的定向固定。该策略包括将11-生物素-dUTP酶促掺入质粒，并使用碱进行链特异性洗脱。固定化的单链DNA用作测序反应的模板，所得标记的寡核苷酸通过碱洗脱。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。固相技术可用于引物特异性或延伸特异性标记。还讨论了将该系统用于自动化DNA测序的可能性。
