Development of automated DNA diagnostic system using solid-phase DNA probe.

(1)我们开发了一个自动化DNA诊断系统，该系统由可预处理的自动进样器、梯度仪、化学惰性高压泵、热控制器、流型（紫外、荧光或化学发光）监视器、馏分收集器和计算机组成。并编制了自动化操作所需的仪器部件和软件。(2)建立了从12b到几kb长的DNA探针的高效固化技术。(3)建立了利用引物/连接子对RE酶切DNA片段进行末端标记的方法，建立了荧光标记引物PCR扩增的高灵敏度方法。(4)该系统基于序列特异性热洗脱色谱法（SSTEC法），从SSTEC模式的峰位直接测量DNA的熔化温度（Tm）。测定Tm的准确度是常规光学方法（高显色或低显色法）的10倍左右，测量的标准偏差小于0.1‰。SSTEC分离的分辨率也很高，我们可以很容易地检测到0.2个含量的差异。(5)利用牛SRY基因部分序列作为固相探针，研究了(1)基于DNA序列的Tm差异，(2)基于点突变的Tm差异，(3)基于位置差异的Tm差异。在结果中，我们发现SSTEC方法即使在一个点突变中也可以检测到。(6) SSTEC法也适用于某些DNA聚合酶的保真度分析。在Taq聚合酶中，我们发现随着扩增程度的增加，牛DNA的Tm值逐渐下降。因此，SSTEC方法是临床和基础生物医学领域DNA突变分析的有力工具。

(1) We developed an automated DNA diagnostic system which comosed of pretreatable autosampler, gradientor, chemically inert high pressure pump, thermal controler, flow type (UV,Fluorescence, or Chemo-luminescence) monitors, fraction collector, and computer. And we prepared some instrumental parts and softweare for automating operation. (2) We establised a high efficient solidification technology for DNA probes from 12b upto several kb long. (3) We developed a terminal labeling method of RE digested DNA fragments using primer/linker, and established higer sensitive method for PCR amplification of fluorescent labeled primer. (4) This system is based on sequence-specific thermal-elution chromatography (SSTEC method), the melting temperature (Tm) of DNA is directly measured from the peak position of SSTEC pattern. The accuracy of Tm measurement was approximetely 10 fold higher than that of conventional optical method (hyperchoromicity or hypochromicity mesurement), and the standard deviation of the Tm measurement was less than 0.1ﾟC.The resolution of SSTEC separation was also high, we can easily detect Tm difference of 0.2ﾟC.(5) By using a partial sequence of bovine SRY gene as a solid-phase probe, we examined (1) Tm difference based on DNA sequence, (2) Tm difference based on point mutation, (3) Tm difference based on positional difference. In the results, we found that the SSTEC method was detectable even in one point mutation. (6) SSTEC method was also applicable in fidelity analysis of some kind of DNA polymerases. In Taq polymerase, we found gradual decline in Tm value of bovine DNA acompanied with degree of amplification on PCR reaction. Therefore, it is concluded that the SSTEC method is useful and powerful tool of mutational analysis of DNA in clinical and basical Biomedical fields.