Semi-quantitative discrimination of HBV mutants using allele-specific oligonucleotide hybridization with Handy Bio-Strand.

The analysis of hepatitis B virus (HBV) mutations is important for understanding HBV progression and for deciding on appropriate clinical treatments. However, it is difficult to determine the quantitative abundance of various mutants in heterogeneous mixtures by conventional methods such as direct sequencing or the TaqMan assay. In this study, we investigated the possibility of using both allele-specific oligonucleotide hybridization (ASOH) and allele-specific oligonucleotide competitive hybridization (ASOCH) with the Handy Bio-Strand system for the quantitative identification of three well-defined HBV variants: the basal core promoter (BCP) mutations (nt1762 and nt1764), the pre-core (PC) mutation (nt1896), and variance at nt1858. Using standardized mixtures of wild-type and mutant DNA, optimal hybridization conditions for ASOH and ASOCH were determined. Next, the performance of these methods was evaluated using actual serum DNAs from HBV patients. Excellent reproducibility was obtained both in the analysis of internal positive controls and in the semi-quantitative categorization of heterogeneous viral mixtures into five abundance groups (0%, 25%, 50%, 75%, and 100% mutant virus). Combined with real-time PCR to determine the HBV viral load, this hybridization method offers a new tool with applications both in HBV clinical research and treatment.

Noncovalent immobilization of streptavidin on in vitro- and in vivo-biotinylated bacterial magnetic particles.

Biotinylated magnetic nanoparticles were constructed by displaying biotin acceptor peptide (BAP) or biotin carboxyl carrier protein (BCCP) on the surface of bacterial magnetic particles (BacMPs) synthesized by Magnetospirillum magneticum AMB-1. BAP-displaying BacMPs (BAP-BacMPs) were extracted from bacterial cells and incubated with biotin and Escherichia coli biotin ligase. Then the in vitro biotinylation of BAP-BacMPs was confirmed using alkaline phosphatase-labeled antibiotin antibody. In contrast, BacMPs displaying the intact 149 residues of AMB-1 BCCP (BCCP-BacMPs) and displaying the COOH-terminal 78 residues of BCCP (BCCP78-BacMPs) were biotinylated in AMB-1 cells. The in vivo biotinylation of BCCP-BacMPs and BCCP78-BacMPs was thought to be performed by endogenous AMB-1 biotin ligase. Streptavidin was introduced onto biotinylated BacMPs by simple mixing. In an analysis using tetramethyl rhodamine isocyanate-labeled streptavidin, approximately 15 streptavidin molecules were shown to be immobilized on a single BCCP-BacMP. Furthermore, gold nanoparticle-BacMP composites were constructed via the biotin-streptavidin interaction. The conjugation system developed in this work provides a simple, low-cost method for producing biotin- or streptavidin-labeled magnetic nanoparticles. Various functional materials can be site selectively immobilized on these specially designed BacMPs. By combining the site-selective biotinylation technology and the protein display technology, more innovative and attractive magnetic nanomaterials can be constructed.

Development of the Handy Bio-Strand and its application to genotyping of OPRM1 (A118G).

We previously developed a three-dimensional microarray system, the Bio-Strand, which exhibits advantages in automated DNA analysis in combination with our Magtration Technology. In the current study, we have developed a compact system for the Bio-Strand, the Handy Bio-Strand, which consists of several tools for the preparation of Bio-Strand Tip, hybridization, and detection. Using the Handy Bio-Strand, we performed single nucleotide polymorphism (SNP) genotyping of OPRM1 (A118G) by allele-specific oligonucleotide competitive hybridization (ASOCH). DNA fragments containing SNP sites were amplified from genomic DNA by PCR and then were fixed on a microporous nylon thread. Thus, prepared Bio-Strand Tip was hybridized with allele-specific Cy5 probes (<15mer), on which the SNP site was designed to be located in the center. By optimizing the amount of competitors, the selectivity of Cy5 probes increased without a drastic signal decrease. OPRM1 (A118G) genotypes of 23 human genomes prepared from whole blood samples were determined by ASOCH using the Handy Bio-Strand. The results were perfectly consistent with those determined by PCR direct sequencing. ASOCH using the Handy Bio-Strand would be a very simple and reliable method for SNP genotyping for small laboratories and hospitals.

Development of an automation system for single nucleotide polymorphisms genotyping using bio-strand, a new three-dimensional microarray.

Previously, we developed a novel three-dimensional microarray system called Bio-Strand, which may be used in various applications including single nucleotide polymorphisms genotyping. In Bio-Strand, samples for detection are immobilized on a one-dimensional thread, which is wound around a cylinder-shaped core to form a three-dimensional thread-and-core structure. The thread-and-core structure is then inserted into a plastic pipette tip, where hybridization and detection are performed. In this study, we have developed an automation system, NIAGALA Bio-Station SDx, which enables automated hybridization and detection during the genotyping procedure using Bio-Strand. Using this system, we have performed the single nucleotide polymorphism (SNP) genotyping of CYP2C, one of the important human cytochrome P450 genes and the results were completely consistent with the genotyping results determined by the TaqMan method.