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Kazunori Okano

Publications and source records attributed to Kazunori Okano.

10 recordsLinked to original sources
PubMed2005

Signal sequence and keyword trap in silico for selection of full-length human cDNAs encoding secretion or membrane proteins from oligo-capped cDNA libraries.

We have developed an in silico method of selection of human full-length cDNAs encoding secretion or membrane proteins from oligo-capped cDNA libraries. Fullness rates were increased to about 80% by combination of the oligo-capping method and ATGpr, software for prediction of translation start point and the coding potential. Then, using 5'-end single-pass sequences, cDNAs having the signal sequence were selected by PSORT ('signal sequence trap'). We also applied 'secretion or membrane protein-related keyword trap' based on the result of BLAST search against the SWISS-PROT database for the cDNAs which could not be selected by PSORT. Using the above procedures, 789 cDNAs were primarily selected and subjected to full-length sequencing, and 334 of these cDNAs were finally selected as novel. Most of the cDNAs (295 cDNAs: 88.3%) were predicted to encode secretion or membrane proteins. In particular, 165(80.5%) of the 205 cDNAs selected by PSORT were predicted to have signal sequences, while 70 (54.2%) of the 129 cDNAs selected by 'keyword trap' preserved the secretion or membrane protein-related keywords. Many important cDNAs were obtained, including transporters, receptors, and ligands, involved in significant cellular functions. Thus, an efficient method of selecting secretion or membrane protein-encoding cDNAs was developed by combining the above four procedures.

5' Flanking Region↗
PubMed2004

A gel-free SNP genotyping method: bioluminometric assay coupled with modified primer extension reactions (BAMPER) directly from double-stranded PCR products.

Inexpensive, high-throughput genotyping methods are needed for analyzing human genetic variations. We have successfully applied the regular bioluminometric assay coupled with modified primer extension reactions (BAMPER) method to single-nucleotide polymorphism (SNP) typing as well as the allele frequency determination for various SNPs. This method includes the production of single-strand target DNA from a genome and a primer extension reaction coupled with inorganic pyrophosphate (PPi) detection by a bioluminometric assay. It is an efficient way to get accurate allele frequencies for various SNPs, while single-strand DNA preparation is labor intensive. The procedure can be simplified in the typing of SNPs. We demonstrate that a modified BAMPER method in which we need not prepare a single-strand DNA can be carried out in one tube. A PCR product is directly used as a template for SNP typing in the new BAMPER method. Generally, tremendous amounts of PPi are produced in a PCR process, as well as many residual dNTPs, and residual PCR primers remain in the PCR products, which cause a large background signal in a bioluminometric assay. Here, shrimp alkaline phosphatase (SAP) and E. coli exonuclease I were used to degrade these components prior to BAMPER detection. The specific primer extension reactions in BAMPER were carried out under thermocycle conditions. The primers were extended to produce large amounts of PPi only when their bases at 3'-termini were complementary to the target. The extension products, PPis, were converted to ATP to be analyzed using the luciferin-luciferase detection system. We successfully demonstrated that PCR products can be directly genotyped by BAMPER in one tube for SNPs with various GC contents. As all reactions can be carried out in a single tube, the method will be useful for realizing a fully automated genotyping system.

Alleles↗
PubMed2003

Microchip electrophoresis of tagged probes incorporated with one-colored ddNTP for analyzing single-nucleotide polymorphisms.

We demonstrate a simple and rapid method for SNP typing, allele frequency determination, and trace mutant analysis that works with even an inexpensive detection system. This method is based on microchip electrophoresis of tagged probes incorporated with one-colored ddNTP (METPOC). The assay uses dye terminator incorporation into a pair of probes of different lengths specific to wild- and mutant-type targets, respectively. They are hybridized to the targets prior to ddNTP-Cy-5 incorporation, which occurs only for a matched probe-target duplex. Because the extension reactions for the two probes are carried out simultaneously in one tube and the products from both probes are analyzed in one channel by one-color fluorescence detection, an accurate comparative analysis of SNPs is possible. SNP typing as well as allele frequency determination in the range above 0.1% can easily be carried out using a commercial microchip electrophoresis system in a few minutes.

Alleles↗
PubMed2003

Automated bead alignment apparatus using a single bead capturing technique for fabrication of a miniaturized bead-based DNA probe array.

We have developed an automated bead alignment apparatus for fabricating a bead-based DNA probe array inside a capillary. The apparatus uses 16 micro vacuum tweezers to extract single beads from among a large amount of beads in bead stock wells. It then manipulates single beads into the probe array capillaries. Single 100-microm-diameter beads were successfully extracted from the water-contained bead-stock well by the vacuum tweezers, which have inner and outer diameters of 50 and 150 microm. An interesting aspect is that unexpected extra beads adsorbed on the outer wall of the vacuum tweezers can be removed using the surface tension force between the water and the atmosphere. In testing the total performance of this apparatus, the DNA probe arrays with 10 sets of probe-conjugated beads and 2 plain beads were produced in the intended order in the capillaries. The time needed to align the 12 beads was 10 min, and the 16 bead arrays were fabricated simultaneously. After hybridization experiments using these fabricated DNA probe arrays, fluorescence from each bead was clearly observed.

Base Sequence↗
PubMed2003

A bead-alignment device with a bead-sized microchamber on a rotating cylinder for fabrication of a miniaturized probe array.

We have developed a compact bead-alignment device with a bead-sized microchamber on a rotating cylinder. The cylinder fits inside a tube with bead-stock pipes containing different probe-conjugated beads and holes for bead-alignment capillaries. The cylinder rotates in the tube, and the microchamber transfers a single 100-microm-diameter bead from a pipe to one of the capillaries in 10 s. By using this process repeatedly, 'bead arrays', which are miniaturized DNA probe arrays in capillaries, were successfully fabricated.

Journal Article↗
PubMed2002

DNA probes on beads arrayed in a capillary, 'Bead-array', exhibited high hybridization performance.

A DNA analysis platform called 'Bead-array' is presented and its features when used in hybridization detection are shown. In 'Bead-array', beads of 100- micro m diameter are lined in a determined order in a capillary. Each bead is conjugated with DNA probes, and can be identified by its order in the capillary. This probe array is easily produced by just arraying beads conjugated with probes into the capillary in a fixed order. The hybridization is also easily completed by introducing samples (1-300 micro l) into the capillary with reciprocal flow. For hybridization detection, as little as 1 amol of fluorescent-labeled oligo DNA was detected. The hybridization reaction was completed in 1 min irrespective of the amount of target DNA. When the number of target molecules was smaller than that of probe molecules on the bead, 10 fmol, almost all targets were captured on the bead. 'Bead-array' enables reliable and reproducible measurement of the target quantity. This rapid and sensitive platform seems very promising for various genetic testing tasks.

Base Sequence↗
PubMed2002

Application of differential display to identify genes for lung cancer detection in peripheral blood.

A blood assay for detection of lung cancer biomarkers could significantly improve cancer patient prognosis and survival rates. Amplified fragment length polymorphism-differential display (AFLP-DD) was used to identify gene transcripts found in lung cancer tissue and the peripheral blood of lung cancer patients. The clones were evaluated for gene expression in lung cancer tissue, peripheral blood of lung cancer patients and healthy volunteers' blood. The isolated gene transcript clones were found to be from the syndecan 1 gene, collagen 1 gene and 2 novel genes. All 4 transcripts were expressed in normal lung tissue, 4 cultured primary lung cells and 6 lung cancer cell lines. RNA was isolated from peripheral blood samples of 69 lung cancer patients. Reverse transcriptase polymerase chain reaction (RT-PCR) was used to test for the presence of cytokeratin 19 and the 4 gene mRNA transcripts in blood RNA. The positive detection rate of at least 1 of the 5 transcripts was 79% for lung adenocarcinoma and 62% for squamous carcinoma. Using RT-PCR, at least 1 of the markers was found in 53% of stage I patients, 100% of stage II, 71% of stage III and 81% of stage IV lung cancer patients. Blood samples from 20 healthy volunteers were also tested, but only 1 of the 5 transcripts was found in 1 patient. These new molecular markers may aid early detection, staging and follow-up of lung cancer patients by RNA isolated from blood.

Adult↗
PubMed2002

Real-time detection of PCR products for comparative analysis of expressed genes using module-shuffling TaqMan probes (MTPs).

A method for comparative analysis of gene expression was developed. It is based on competitive PCR amplification and real-time detection of PCR products and it uses module-shuffling sequences as "universal TaqMan probes". Namely, cDNA-tagged module-shuffling sequences, which derived from different sources, were amplified in one reaction tube by the same primer set. Two kinds of fluorescent TaqMan probes with different module-shuffling sequences (MTPs) detect their own targets. The method can detect different amounts of expressed genes derived from different sources; accordingly, it was successfully used for comparative analysis of expressed mouse genes.

Base Sequence↗
PubMed2002

Rapid multiplex single nucleotide polymorphism genotyping based on single base extension reactions and color-coded beads.

A single nucleotide polymorphism (SNP) typing method using color-coded beads is promising because it is easy to use and inexpensive. However, the present protocols are not suitable for clinical and diagnostic applications because they need centrifugation for bead-washing. Here, we developed a simplified protocol without a bead-washing procedure that enables SNP typing of PCR amplified fragments in only 30 min.

Journal Article↗