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

(1)我们研制了一种自动化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、温控仪、流式（紫外、荧光或化学发光）监测仪、馏分收集器和计算机组成。并为自动化操作准备了部分仪器部件和软件。(2)我们建立了一种从12 b到几kb长度的DNA探针的高效固化技术。(3)建立了一种用引物/接头对RE酶切的DNA片段进行末端标记的方法，并建立了灵敏度更高的荧光标记引物PCR扩增方法。(4)该系统基于序列特异性热洗脱色谱（SSTEC方法），从SSTEC图谱的峰位直接测量DNA的解链温度（Tm）。Tm测量的准确度比传统的光学方法（增色法或减色法）提高了近10倍，Tm测量的标准偏差小于0.1 μ C，SSTEC分离的分辨率也很高，可以很容易地检测到0.2 μ C的Tm差异。(5)以牛SRY基因的部分序列为固相探针，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是可检测的。(6)SSTEC方法也适用于某些DNA聚合酶的保真度分析。在Taq聚合酶中，随着PCR反应的扩增程度，牛DNA的Tm值逐渐下降。因此，SSTEC方法在临床和基础生物医学领域是DNA突变分析的有效工具。

(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.