Double stranded DNA sequencing as a choice for DNA sequencing.
Double stranded DNA sequencing as a choice for DNA sequencing.
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双链 DNA 测序作为 DNA 测序的一种选择。
DOI：
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发表时间：
1988
影响因子：
14.9
通讯作者：
Chris Somerville
中科院分区：
文献类型：
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作者：
Hong Zhang;Randy Scholl;J. Browse;Chris Somerville
双链DNA测序（1）因其简单、方便而受到青睐。然而，直到最近，这种方法的质量才变得与单链DNA测序相当。我们认为有两个因素限制了双链DNA测序的普及：1，模板DNA的质量; 2，固有的性质，DNA聚合酶。修饰的T7 DNA聚合酶（测序酶）具有几种特性，使其更适合测序（2）。在双链DNA测序中，我们用测序酶取代了Klenow聚合酶I，并且通过仔细注意用于变性和回收模板DNA的条件，我们现在常规地产生与单链DNA测序一样好的测序结果（见图）。在这里，我们提出了一个逐步的协议，为模板DNA的碱性变性，我们的测序反应的建议。
Double stranded DNA sequencing (1) is favored because of its simplicity, and convenice. However it is only recently that the quality of this method has become comparable with single stranded DNA sequencing. We believe that two factors have limited the popularity of double stranded DNA sequencing: 1, the quality of the template DNA; 2, the inherent propertv.,of the DNA polymerase. The modified T7 DNA polymerase (Sequenase ) has several properties which make it more suitable for sequencing (2). We replaced the Klenow Polymerase I with Sequenase in our double stranded DNA sequencing, and by paying careful attention to the conditions used to denature and recover the template DNA, we are now routinely producing sequencing results which are as good as single stranded DNA sequencing (see figure). Here, we present a step-by-step protocol for the alkaline denaturation of template DNA, and our recommendations for the sequencing reaction.