Hattori M, Sakaki Y
Anal Biochem. 1986 Feb 1;152(2):232-8. doi: 10.1016/0003-2697(86)90403-3.
The dideoxy sequencing method in which denatured plasmid DNA is used as a template was improved. The method is simple and rapid: the recombinant plasmid DNA is extracted and purified by rapid alkaline lysis followed by ribonuclease treatment. The plasmid DNA is then immediately denatured with alkali and subjected to a sequencing reaction utilizing synthetic oligonucleotide primers. It takes only several hours from the start of the plasmid extraction to the end of the sequencing reaction. We examined each step of the procedure, and several points were found to be crucial for making the method reproducible and powerful: (i) the plasmid DNA should be free from RNA and open circular (or linear) DNA; (ii) a heptadecamer rather than a pentadecamer is recommended as a primer; and (iii) the sequencing reaction should be done at 37 degrees C or higher rather than at room temperature. The method enabled us to determine the sequence of more than a thousand nucleotides from a single template DNA.
以变性质粒DNA为模板的双脱氧测序方法得到了改进。该方法简单快速：重组质粒DNA通过快速碱裂解法提取纯化，随后进行核糖核酸酶处理。然后，质粒DNA立即用碱变性，并利用合成寡核苷酸引物进行测序反应。从开始提取质粒到测序反应结束仅需几个小时。我们检查了该过程的每一步，发现有几点对于使该方法具有可重复性和高效性至关重要：（i）质粒DNA应不含RNA和开环（或线性）DNA；（ii）推荐使用十七聚体而非十五聚体作为引物；（iii）测序反应应在37℃或更高温度下进行，而不是在室温下。该方法使我们能够从单个模板DNA中确定一千多个核苷酸的序列。
