Taq DNA聚合酶具有反应速度快、温度作用范围广及良好的续进性等特点,可视为一种理想的DNA顺序分析酶。本文首先对非对称性PCR扩增过程中单、双链DNA产物的积累情况进行了分析,然后采用标记延伸二步法,对Taq DNA聚合酶的性质及影响因素进行分析。为进一步改进Taq DNA聚合酶测序的方法,本反应建立了“Klenow-型”的直接掺入标记同位素测序法,即在反应液中加入与标记核苷酸相应的一定浓度的冷dNTP。此法不但解决了二步法中引物后部分DNA顺序无法读出的缺点,而且简化了反应步骤,亦能得到令人满意的顺序分析结果,每次可读出至少400碱基的序列。

Taq DNA polymerase is an ideal enzyme for DNA sequencing because it's fast, highly processive and active over a broad range of temperature. Here, we have studied the conditions for relative accumulation of dsDNA and ssDNA in asymmetic PCR amplification. The PCR product was sequenced directly with Taq DNA polymerase by two-step labeling-extension. protocol. In order to improve the "Klenow-type" protocol for incorporation of labeled nucleotide during the sequencing reaction, in which 3.0μmol/L cold dCTP were added when [α-32p] dCTP was used for direct sequencing of PCR products. This method solved the problem that the parts of sequencing ladders adjacent the primer could not be read with two-step protocol and labeling step was also omitted. The satisfactory sequencing ladders have been obtained by this improved "Klenow-type" protocol.