生物晶片或微陣列是在固態基材上以微小化技術固定數量極多的不同核酸或蛋白質探針，可應用雜合原理與方法，經由檢測而獲取大量有關探針或標的物樣品的資訊。我們擬開發一套命名為非均相雜和反應及非均相聚合連鎖反應(HHHM)微陣列系統。此微陣列系統之核心技術為利用磁珠做為固定探針之固態基材，使液態雜和反應可在微井中進行以改善微陣列雜和反應及靈敏度、且應用範圍較廣泛。在此研究中，我們發展將核酸引子固定於磁珠固態基材的方法，並利用結合核酸引子的磁珠以非均相聚合鏈鎖反應將核酸探針倍增之方法。經由雜和反應後，我們發現PolyScience®公司的氨基磁珠有最好的核酸-核酸雜和反應效率。而且，我們以人類血清白蛋白(HSA)及anti-HSA抗體為例子，發展將蛋白質固定於磁珠固態基材的方法。經由雜和反應後，PolyScience®公司的羧基磁珠結合HSA蛋白質或anti-HSA抗體之可檢測螢光標識標的物最低含量分別為0.04及0.004微克。最後，我們以人類非小型肺癌細胞株(H460)之核蛋白為例子，發展核酸-蛋白質結合反應的實驗方法。
當酵母菌處於低濃度葡萄糖環境下，高親和性己糖轉運蛋白(HXT6, HXT7)基因已知將會被誘發並且大量表現。在此研究中，我們藉由利用高親和性己糖轉運蛋白啟動子所啟動之轉化酶(invertase)表現可以幫助回復酵母菌菌株GN 3C.2於棉子糖(raffinose)培養基中的生長缺陷，並且發現HXT7啟動子相較於HXT6啟動子具有更強及維持轉化酶高表現量的能力，進一步的比較發現，HXT7啟動子所表現之轉化酶基本表現量分別是酵母菌菌株GN 3C.2及W303-1之內源性轉化酶的139及28倍。另外，經由與熟知的持續表現型酒精去氫酶啟動子(constitutive ADH1 promoter)比較，HXT7啟動子也可以於短時間內，表現出高量的增強綠螢光蛋白(enhanced green fluorescence protein)，因此，HXT7啟動子具有在酵母菌菌株中做為建構異源(heterologous)蛋白質表現系統的潛力。另一方面由文獻得知，在缺乏葡萄糖的環境下轉錄因子Rgt1會抑制己糖轉運蛋白的表現，我們發現長度1.25 kb的HXT7啟動子包含有三個Rgt1結合位置，其活性會因為將Rgt1基因刪除後而增加三倍，並且在缺乏葡萄糖的環境下只有一個位於-1164至-1158區間之Rgt1結合位置參與HXT7啟動子之調控。

Biochips or microarrays are microscopic arrays of large set of DNA sequences or peptides immobilized on solid substrates. They are, in principle, hybridization-based methods and are useful for obtaining information about either the probes or the target molecules. We intend to develop a novel microarray system, designated as HHHM (Heterogeneous-phase Hybridization and Heterogeneous-phase Magnetic-PCR). The core technology of this HHHM system is using magnetic bead as solid support for probe immobilization. It performs the liquid phase hybridization reaction in microwells to optimize array hybridization and sensitivity and to extend their range of applications. In this study, we developed methods for immobilizing DNA primer on magnetic bead and for PCR amplification of DNA probes using the magnetic bead conjugated primer as one of the two primers. After hybridization reaction, we found the PolyScience® amine magnetic bead displayed the best efficiency of DNA-DNA hybridization. Furthermore, we used the human serum albumin (HSA) and anti-HSA antibodies as examples to develop methods for immobilizing protein on magnetic bead. The minimal amount of fluorescence labeled targets was 40 and 4 ng, respectively, detected by the PolyScience® carboxylated magnetic bead conjugated with HSA or anti-HSA antibodies. Finally, we used the nuclear protein extracted from human non-small cell lung cancer cells (H460) as an example to develop a protocol for detecting double-strand DNA-protein interaction.

In Saccharomyces cerevisiae, both the high-affinity hexose transporter genes HXT6 and HXT7 are known to be induced and highly expressed under low hexose concentration. In this work we report that both the high-affinity hexose transporter HXT6 and HXT7 promoters are sufficient for restoring the growth rate of S. cerevisiae strain GN 3C.2 by complementary expression of invertase under raffinose medium. Moreover, we found that HXT7 promoter was more powerful than HXT6 one to produce and maintain high level invertase. Surprisingly, HXT7 promoter also displayed a high basal expression level of invertase for about 139- and 28-fold more than those of strains GN 3C.2 and W303-1, respectively. In addition, HXT7 promoter expressed relative higher level of enhanced green fluorescent protein (eGFP) than that of constitutive ADH1 one in three different S. cerevisiae strains, even during short-term incubation in glucose medium. Therefore, HXT7 promoter has the potential application for heterologous protein expression in S. cerevisiae. On the other hand, the Rgt1 transcription factor was reported as a repressor for HXT induction in the absence of glucose. We found the activity of the 1.25 kb HXT7 promoter containing three Rgt1 binding sites was upregulated for about 3-fold when the RGT1 gene was deleted. And only the Rgt1 binding site located at -1164 to -1158 was involved in the regulation of HXT7 promoter in the absence of glucose.

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