Ansorge W, Sproat B S, Stegemann J, Schwager C
J Biochem Biophys Methods. 1986 Dec;13(6):315-23. doi: 10.1016/0165-022x(86)90038-2.
A method and instrument for automated DNA sequencing without radioactivity have been developed. In spite of the success with radioactive labels there are drawbacks attached to the technique, such as hazards in the handling, storage and disposal of radioactive materials, and the considerable cost of the radiolabelled nucleoside triphosphates. In addition, there is deterioration of sample quality with time. A sulphydryl containing M13 sequencing primer has been synthesised and subsequently conjugated with tetramethylrhodamine iodoacetamide. The fluorescent primer is used to generate a nested set of fluorescent DNA fragments. The fluorescent bands are excited by a laser and detected in the gel (detection limit about 0.1 fmol per band) during electrophoresis, and sequence data from the four tracks are transferred directly into a computer. Standard gels, 200 mm wide with 20 sample slots have also been used. The device contains no moving parts. At present 250-300 bases can be read in 6 h. The system is capable of single base resolution at a fragment length of at least 400 bases.
已经开发出一种无放射性的自动化DNA测序方法及仪器。尽管放射性标记取得了成功，但该技术仍存在一些缺点，如放射性材料在处理、储存和处置方面的危害，以及放射性标记的核苷三磷酸的高昂成本。此外，样品质量会随时间下降。已经合成了一种含巯基的M13测序引物，并随后与四甲基罗丹明碘乙酰胺偶联。荧光引物用于生成一组嵌套的荧光DNA片段。荧光带在电泳过程中由激光激发，并在凝胶中检测（每条带的检测限约为0.1飞摩尔），来自四条泳道的序列数据直接传输到计算机中。也使用了200毫米宽、有20个样品槽的标准凝胶。该装置没有活动部件。目前，在6小时内可以读取250 - 300个碱基。该系统能够在至少400个碱基的片段长度上实现单碱基分辨率。
