Parvin J D, Smith F I, Palese P
DNA. 1986 Apr;5(2):167-71. doi: 10.1089/dna.1986.5.167.
A simple and efficient nucleic acid sequencing method is described in which RNA transcription by the SP6 polymerase is specifically terminated using 3'-deoxynucleotide triphosphates. Initial difficulties in resolving the RNA ladder were overcome by replacing guanosine triphosphate by inosine triphosphate in the reaction mixture and electrophoresing gels at high temperature (50 degrees C). This method presents advantages over current sequencing techniques: Unprocessed plasmid DNA is the template and preparation of inserts and/or single-stranded templates is unnecessary. Use of the specific promoter for SP6 polymerase removes the need for a primer in sequencing reactions.
本文描述了一种简单高效的核酸测序方法，其中通过使用3'-脱氧三磷酸核苷酸特异性终止SP6聚合酶介导的RNA转录。通过在反应混合物中用三磷酸肌苷替代三磷酸鸟苷，并在高温（50℃）下进行凝胶电泳，克服了最初解析RNA梯状条带的困难。该方法相对于当前的测序技术具有以下优点：未加工的质粒DNA作为模板，无需制备插入片段和/或单链模板。使用SP6聚合酶的特异性启动子消除了测序反应中对引物的需求。
