A NONRADIOACTIVE AUTOMATED-METHOD FOR DNA-SEQUENCE DETERMINATION
A NONRADIOACTIVE AUTOMATED-METHOD FOR DNA-SEQUENCE DETERMINATION
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DOI：
10.1016/0165-022x(86)90038-2
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发表时间：
1986-12-01
影响因子：
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通讯作者：
SCHWAGER, C
中科院分区：
文献类型：
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作者：
ANSORGE, W;SPROAT, BS;SCHWAGER, C
研制了一种无放射性的DNA自动测序方法和仪器。尽管放射性标记取得了成功，但该技术也存在缺陷，例如放射性材料的处理、储存和处置中的危险，以及放射性标记的核苷三磷酸的相当大的成本。此外，随着时间的推移，样品质量也会下降。合成了含巯基的M13测序引物，随后与四甲基罗丹明碘乙酰胺缀合。荧光引物用于产生一组嵌套的荧光DNA片段。在电泳过程中，荧光带由激光激发并在凝胶中检测（检测限约为0.1 fmol/带），来自四个轨道的序列数据直接传输到计算机中。还使用了具有20个样品槽的200 mm宽的标准凝胶。该器械不含活动部件。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个碱基的片段长度下进行单碱基解析。
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 synthesized 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.