Abstract
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 PolyScienceR 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 PolyScienceR 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.