高温反向顺序测定

Reverse DNA Sequencing by Using the Heat-stable Bst Polymerase System

单链克隆DNA的反向测序是一个简单和快速的方法,这个方法可利用原来的DNA再进行反向顺序测定,从而达到校正和积累DNA数据的作用。由于反向测序的本质是双链测序,所得到的DNA顺序的图谱背景较深,伴有杂带,或在X光显影上是不清晰的。本文报道利用我们实验室所开发的热稳定性的Bst聚合酶系统,进行反向测序克服了这些缺点,获得的图谱可以和单链DNA模板测序的图谱相媲美。我们以nod D 487-mp 8 ss-DNA为模板进行反向测序,证明Bst聚合酶在65℃反应能得到满意的结果。

Reverse DNA sequencing was a simple and rapid method to check the available DNA data by sequencing the original DNA in the opposite orientation. Because this method has the double stranded sequencing nature, high background, extra bands occurred not unfrequently on the autoradiographs. These short comings have now been overcome by using the heat stable Bst polymerase system developed in this lab. The reverse DNA sequence patterns obtained by using this system were now fully comparable with those patterns generated on the single-stranded DNA template.