DIDEOXY SEQUENCING METHOD USING DENATURED PLASMID TEMPLATES
DIDEOXY SEQUENCING METHOD USING DENATURED PLASMID TEMPLATES
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DOI：
10.1016/0003-2697(86)90403-3
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发表时间：
1986-02-01
影响因子：
2.9
通讯作者：
SAKAKI, Y
中科院分区：
文献类型：
--
作者：
HATTORI, M;SAKAKI, Y
对以变性质粒DNA为模板的双脱氧测序方法进行了改进。该方法简单、快速：通过快速碱裂解、核糖核酸酶处理提取和纯化重组质粒DNA。然后立即用碱使质粒 DNA 变性，并利用合成寡核苷酸引物进行测序反应。从质粒提取开始到测序反应结束仅需几个小时。我们检查了该程序的每个步骤，发现有几点对于使该方法具有可重复性和强大的作用至关重要：（i）质粒 DNA 应该不含 RNA 和开放环状（或线性）DNA； (ii) 建议使用十七聚体而不是十五聚体作为底漆； (iii)测序反应应在37℃下进行。 C 或更高，而不是在室温下。该方法使我们能够从单个 DNA 模板中确定超过 1000 个核苷酸的序列。
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.degree. 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.