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

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(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.