1,720,991 research outputs found
Mismatch DNA-specific enzymatic cleavage employed in a new method for the electrochemical detection of genetic mutations
No full textUtilizing enzymatic mismatched DNA-specific cleavage and electrocatalytic signaling, a new electrochemical method for the detection of DNA mutations was developed and successfully applied to detect various mutations in the BRCA1 gene.This work was supported by the Brain Korea 21 (BK21)
program and the Centre for Ultramicrochemical Process
Systems
SNPs detection by a single-strand specific nuclease on a PNA zip-code microarray
No full textIn this report, a reliable peptide nucleic acid (PNA) microarray-based method for accurately detecting single nucleotide polymorphism (SNP) in human genes is described. The technique relies on the mismatched cleavage activity of a single-strand specific (SSS) nuclease. PCR amplification was performed to prepare gene fragments containing the mutation sites. The amplified fragments were then employed as templates for the SSS nuclease reaction using chimeric probes, modified with biotin at the Tend and extended with a unique anchoring zip-code complement sequence at the Tend. The SSS nuclease promotes cleavage of heteroduplex DNAs at base mismatched positions to produce crumbled chimeric probes in the presence of imperfectly matching template strands. in contrast, the probes remain intact when they interact with perfectly matched template strands. Only the non-fragmented probes generate fluorescence signals after treatment with streptavidin-Cy3 on the PNA zip-code array. This methodology was used to successfully genotype selected Korean-specific BRCA mutation sites with wild type and mutant samples. The investigation has led to the development of a reliable SSS nuclease-based system for the diagnosis of human genetic mutations or SNPs. (C) 2008 Elsevier B.V. All rights reserved.Thisworkwas supported by the Brain Korea 21 (BK21) program,
the Korea Research Foundation Grant funded by the Korean Government
(MOEHRD) (KRF-2006-331-D00114) and the Centre for
Ultramicrochemical Process Systems
A New Non-Isolated High Step-up Converter with Reduced Voltage Stress
No full textThis paper presents non-isolated high step-up converters with reduced voltage stress. Based on the conventional flyback converter, a high step-up converter is derived. The voltage stresses of the switch and diodes are limited by using a clamping diode and voltage doubler structure. Also, to further reduce the voltage stresses of them, another high step-up converter is proposed. The proposed converter has one additional capacitor to serve as a doubler capacitor. By using this capacitor, the voltage stresses of the switch and diodes can be clamped to the lower voltage from the boost converter. The operational principle, analysis and design considerations of the proposed converter are presented in this paper. The validity of this study is confirmed by the experimental results from 24 V input and 250 V/125 W output prototype
Microarray-based detection of Korean-specific BRCA1 mutations
No full textA reliable multiplex assay procedure to detect human genetic mutations in the breast cancer susceptibility gene BRCA1 using zip-code microarrays and single base extension (SBE) reactions is described. Multiplex PCR amplification was performed to amplify the genomic regions containing the mutation sites. The PCR products were then employed as templates in subsequent multiplex SBE reactions using bifunctional primers carrying a unique complementary zip sequence in addition to a mutation-site-specific sequence. The SBE primers, terminating one base before their mutation sites, were extended by a single base at a mutation site with a corresponding biotin-labeled ddNTP. Hybridization of the SBE products to zip-code microarrays was followed by staining with streptavidin-Cy3, leading to successful genotyping of several selected BRCA1 mutation sites with wild-type and heterozygote mutant samples from breast cancer patients. This work has led to the development of a reliable DNA microarray-based system for the diagnosis of human genetic mutations.This work was supported by the Korea Research
Foundation Grant funded by the Korean Government
(MOEHRD, KRF-2006–331-D00114) and by the Brain Korea 21
(BK21) program and the Center for Ultramicrochemical Process
Systems sponsored by KOSEF
Thiopurine S-methyltransferase polymorphisms and the relationship between the mutant alleles and the adverse effects in systemic lupus erythematosus patients taking azathioprine
No full textObjective. The present study sought to elucidate the genetic basis of thiopurine methyltransferase (TPMT) polymorphism and subsequently to investigate the relationship between mutant TPMT and an adverse response observed in Korean patients with systemic lupus erythematosus (SLE) taking azathioprine (AZA). Methods. The TPMT genotype of 342 patients with SLE was determined by MALDI-TOF mass spectroinetry and correlated with the effects of clinical exposure to AZA. Results. TPMT polymorphism was detected in 17 of the 342 study subjects (5.0%), 12 heterozygous for the TPM-T*3C allele and 5 heterozygous for the TPMT*6 allele. Numerous patients taking AZA demonstrated adverse drug responses. Severe nausea occurred in 4 patients with the TPMT*3C allele, while I patient with the TPMT*6 allele suffered severe bone marrow toxicty Leucopenia (n = 17), nausea (n = 4), and abnormal liver function (n = 1) were suspected in 23 of the 94 lupus patients taking AZA. AZA was relatively well tolerated by the remainder of the patients. The heterozygous genotype for the TPMT*3C and *6 alleles was frequently detected it? Korean SLE patients. Conclusion. Contrary to previous hypotheses, this study identified no statistical correlation between TPMT geno-type and AZA toxicity We thus conclude that TMPT genotyping cannot replace regular blood monitoring in SLE patients receiving AZA treatment
Mismatch DNA-specific enzymatic cleavage employed in a new method for the electrochemical detection of genetic mutations
No full textA Polydiacetylene Microchip Based on a Biotin–Streptavidin Interaction for the Diagnosis of Pathogen Infections
No full textA micropatterned polydiacetylene (PDA) chip utilizing the unique, fluorogenic property of PDA and a specific biotin-streptavidin (STA) interaction, is constructed to detect pathogen infections. To construct the PDA chip, biotin-modified diacetylene liposomes are immobilized on aldehyde glass and conjugated with STA, followed by UV irradiation to polymerize the STA-functionalized diacetylene liposomes. Genomic DNA of a model pathogen, Chlamydia trachomatis, is isolated from human samples and biotin-labeled target DNA is obtained through PCR amplification using biotin-11-dUTP. Owing to the stimulus caused by the biotin-STA interaction, the biotinylated DNA induces an intense fluorescence signal on the immobilized PDA. By using this strategy, it is possible to diagnose Chlamydia infections by applying DNA samples from several nonhealthy humans to a single PDA chip. The results of this study serve as the basis for a new strategy for fluorogenic PDA microarray-based diagnosis of pathogen infections.This work was supported by the Korea Science and Engineering
Foundation (KOSEF) grant funded by the Korea government (MOST; Basic Research Program, No. R01-2007-000-11851-0), R
& BD Project in DAEDEOK INNOPOLIS (M-2007-048), and the
Center for Ultramicrochemical Process Systems (CUPS) sponsored
by KOSEF
Multiplexed Amino Acid Array Utilizing Bioluminescent Escherichia coli Auxotrophs
No full textWe describe a novel multiplex "amino acid array" for simultaneously quantifying different amino acids based on the rapid growth of amino acid auxotrophic E. coli. First, we constructed genetically engineered amino acid auxotrophs of E. coli containing a bioluminescence reporter gene, yielding concomitant luminescence as a response to cell growth, and then immobilized the reporter cells within individual agarose of respective wells in a 96-well plate serving as a mimic of a biochip. Using the amino acid array, we were able to determine quantitatively the concentrations of 16 amino acids in biological fluid by simply measuring bioluminescent signals from the immobilized cells within 4 h without pre- and post-treatment. The clinical utility of this method was verified by quantifying different amino acids in dried blood spot specimens from clinical samples for the diagnosis of metabolic diseases of newborn babies. This method serves as a convenient route to the rapid and simultaneous analysis of multiple amino acids from complex biological fluids and represents a new analytical paradigm that can replace conventional, yet laborious methods currently in use.This work was supported by the grant from Basic Science
Research Program through the National Research Foundation of
Korea (NRF) funded by the Ministry of Education, Science and
Technology (No. 2009-0080602), the Center for Ultramicrochemical
Process Systems by KOSEF, and BK21
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