• Biotechnology and Bioprocess Engineering:BBE
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• v.7 no.5
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• pp.311-315
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• 2002

By the in vitro combinatorial mutagenesis, which is a sequential reaction of PCR mutagenesis and in vitro coupled transcription/translation with Escherichia coli S30 extract, S65 and E222 of green fluorescent protein of Aequarea victoria were comprehensively changed to all possible combinations of amino acids, thus totally 400 mutant (including a wild type) proteins were simultaneously produced and their fluorescent properties were analyzed. Although a few mutations had been reported so far at the 222nd position, replacement E222 to all other19 amino acids gave fluorescent signal to the mutants by changing Ser 65 to Ala together. Among the mutants, replacement to G, A, S, Q, H and C gave relatively high fluorescence. The in vitro combinatorial mutagenesis, therefore, has been proved valuable for comprehensive structure-function studies of proteins.

• Journal of Microbiology and Biotechnology
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• v.22 no.1
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• pp.13-24
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• 2012

The genetic diversity of plant growth-promoting rhizobacterial (PGPR) fluorescent pseudomonads associated with the sugarcane (Saccharum officinarum L.) rhizosphere was analyzed. Selected isolates were screened for plant growthpromoting properties including production of indole acetic acid, phosphate solubilization, denitrification ability, and production of antifungal metabolites. Furthermore, 16S rDNA sequence analysis was performed to identify and differentiate these isolates. Based on 16S rDNA sequence similarity, the isolates were designated as Pseudomonas plecoglossicida, P. fluorescens, P. libaniensis, and P. aeruginosa. Differentiation of isolates belonging to the same group was achieved through different genomic DNA fingerprinting techniques, including randomly amplified polymorphic DNA (RAPD), amplified ribosomal DNA restriction analysis (ARDRA), repetitive extragenic palindromic (REP), enterobacterial repetitive intergenic consensus (ERIC), and bacterial repetitive BOX elements (BOX) analyses. The genetic diversity observed among the isolates and rep-PCR-generated fingerprinting patterns revealed that PGPR fluorescent pseudomonads are associated with the rhizosphere of sugarcane and that P. plecoglossicida is a dominant species. The knowledge obtained herein regarding the genetic and functional diversity of fluorescent pseudomonads associated with the sugarcane rhizosphere is useful for understanding their ecological role and potential utilization in sustainable agriculture.

• Research in Plant Disease
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• v.14 no.3
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• pp.219-222
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• 2008

Real time PCR was used to discriminate Colletotrichum acutatum and C. gloeosporioides for analysis of population density. Two primers, caInt2 and cgint, used for conventional PCR to discriminate two species were modified with fluorescent dye to make probe for real time PCR. Fluorescence signals were successfully detected by fCaInt2 and vCgint probe coupled with primer pair Unicon and Unicor1 resulting in discrimination of C. acutatum and C. gloeosporioides by comparison of delta Rn value.

• Korean Journal of Animal Reproduction
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• v.20 no.3
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• pp.323-331
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• 1996

Predetermination of sex in mammalian species has many aspects of application including the prenatal diagnoses of genetic disorders in humans and sex-selected breeding programs in the animal industry. Embryos sexing can be carried out using the polymerase chain reaction (PCR) to amplify specific sequences present in the sex chromosomes, or by fluorescent in situ hybridization (FISH) of specific probes to the X and Y chromosomes. A 3.3 kb porcine male-specific DNA fragment (pEM39) was cloned previously in our laboratory. In this study, FISH and PCR methods were employed to examine if the pEM39 can be used a sex-specific DNA probes Porcine ovaries were obtained from a local slaughter house and oocytes collected. All oocytes were subjected to in vitro maturation followed by 1n vitro fertilization. Parthenogenetically activated embryos were served as a negative control. Embryonic samples were collected at the 2-cell stages and PCR was performed to analyze DNA. Among 10 embryos examined, four embryos were identified as males and six were females. The cloned male-specific DNA fragment showed male-specificity for the cells in the liver tissue and the porcine early embryos by FISH. It was also demonstrated that the cloned male-specific DNA is localized on the hetero chromatic region of the long arm in the Y chrom-osome (Yq) as shown by the FISH and karyotyping. The results suggest that the cloned male-specific DNA fragment may be useful for predetermination of sex with a few embryonic cells. The porcine male-specific sequence can be a reliable index for embryo sexing by PCR.

• The Plant Pathology Journal
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• v.27 no.4
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• pp.385-389
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• 2011

A new reverse transcription loop-mediated isothermal amplification (RT-LAMP) method for the Potato leafroll virus (PLRV) was developed and compared with conventional reverse transcription polymerase chain reaction (RT-PCR) to address its advantages over RTPCR. RT-LAMP primers were designed from the open reading frame 3 (ORF3) sequence of PLRV. The RT-LAMP reactions were conducted without or with a set of loop primers. By real-time monitoring using Turbimeter, the RT-LAMP (with loop primers) detects PLRV in less than 30 min, compared to 120 min of RT-PCR. By adding fluorescent reagent during the reaction, final products of the RT-LAMP were fluorescently visualized under UV light or could be differentiated by naked-eye inspection under normal light. The RT-LAMP was extremely sensitive, about 2000-fold more sensitive than RT-PCR. This study presents great potential of the RT-LAMP for diagnosis and PLRV epidemiology because RT-LAMP method is speedy, sensitive, inexpensive, and convenient.

• Tuberculosis and Respiratory Diseases
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• v.39 no.1
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• pp.1-6
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• 1992

The diagnosis of tuberculosis is usually established using staining and culturing techniques. Fluorescent stains have improved the sensitivity of direct microscopy. Improved culture media coupled with radiometric means of detecting early mycobacterial growth have shortened the time needed for cultural diagnosis. Rapid immunodiagnostic techniques based on the detection of mycobacterial antigen or of antibodies to theses antigens have not, however, come into widespread clinical use. The DNA or RNA hybridization tests with labeled specific probes which have been described so far are not sensitive enough to be used for clinical speicimens without prior culturing. The advent of the polymerase chain reaction (PCR) has opened new possibilities for diagnosis of microbial infections. This technique has already been applied to a number of microorganisms. In the field of mycobacteria the PCR has been used to identify and to detect DNAs extracted from various mycobacteria. However, despite the extraordinary enthusiasm surrounding this technique and the considerable investiment, PCR has not emerged from the developmental "trenches" in the passed several years. It may be a considerable lenth of time before clinical microbiology laboratories become PCR playgrounds because many details remain to be worked out.

• Annals of Laboratory Medicine
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• v.39 no.1
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• pp.50-57
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• 2019

Background: The Automated Fluorescent Immunoassay System (AFIAS) rotavirus assay (Boditech Med Inc., Chuncheon, Korea) is a new rapid antigen test for rotavirus detection. We evaluated the performance of this assay for detecting rotaviruses and their specific genotypes in clinical stool samples. Methods: AFIAS rotavirus assay was performed in 103 rotavirus-positive and 103 rotavirus-negative stool samples (confirmed by both PCR and ELISA), and its results were compared with those of PCR, ELISA, and immunochromatographic assay (ICA). We evaluated diagnostic sensitivity/specificity, the detectability of rotavirus subtypes, lower limit of detection (LLOD), reproducibility, cross-reactivity, and interference of AFIAS rotavirus assay. Results: Based on PCR and ELISA results, diagnostic sensitivity and specificity of the AFIAS rotavirus assay were both 99.0%. LLOD results showed that the AFIAS assay had sensitivity similar to or greater than ICA and ELISA. High reproducibility was confirmed, and no cross-reactivity or interference was detected. This assay could detect genotypes G1P[8], G2P[4], G3P[8], G4P[6], G4P[8], G8P[4], G8P[8], G9P[4], and G9P[8]. Conclusions: The AFIAS rotavirus assay showed high reproducibility, sensitivity, and specificity as well as excellent agreement with ELISA, PCR, and ICA. It detected the most common as well as unusual genotypes of rotavirus prevalent in Korea. It could be a useful onsite assay for rapid, convenient, and cost-effective detection of rotavirus infection.

• Journal of Genetic Medicine
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• v.4 no.1
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• pp.84-87
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• 2007

Spinocerebellar Ataxia Type 3 (SCA 3) is a rare autosomal dominative disorder in which one of the neurodegenerative disorders is caused by a CAG repeat expansion on chromosome 14q32.1. The age at onset of disease is related to the size of the expanded CAG repeat. We present the prenatal diagnosis of SCA3 in a woman whose husband was known to carry an unstable CAG repeat expansion in the MJD gene. The diagnosis was made using PCR with a fluorescent probe for an expanded MJD allele. The normal ranges of (CAG)n of SCA3 are 14~38 repeats. The husband, who had a family history of SCA 3, has an expanded allele of 69 CAG repeats with a normal allele of 27 repeats. His wife had two normal alleles with 26 and 32 CAG repeats. The fetus had two normal alleles with 26 and 27 CAG repeats; consequently, the baby w as healthy. We report a case of prenatal diagnosis of SCA3 using a fluorescent PCR which is rapid and accurate.

• Journal of Plant Biotechnology
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• v.30 no.4
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• pp.307-313
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• 2003

Fluorescent differential display (FDD) is a method for identifying differentially expressed genes in eukaryotic cells. The mRNA FDD technology works by systematic amplification of the 3' terminal regions of mRNAs. This method involve the reverse transcription using anchored primers designed to bind 5'boundary of the poly A tails, followed by polymerase chain reaction (PCR) amplification with additional upstream primers of arbitrary sequences. The amplified cDNA subpopulations are separated by denaturing polyacrylamide electrophoresis. To identify the genes involved in the development of first trap leaf, we applied a FDD method using mRNAs from leaf base, first trap leaf and flower tissue, respectively. We screened several genes that expressed specifically in first trap leaf. Nucleotide sequence analysis of these genes revealed that these were protease inhibitor (PI), myo-inositol-1-phosphate synthase and lipocalin-type prostaglandin D synthase. Northern blot analysis showed that these genes were expressed specifically in first trap leaf (in vivo and in vitro). FDD could prove to be useful for simultaneous scanning of transcripts from multiple cDNA samples and faster selection of differentially expressed transcripts of interest.

• Journal of Life Science
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• v.27 no.6
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• pp.617-622
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• 2017

In this study, we sought to develop an effective cloning system by which human cytochrome $b_5$ (cyt $b_5$) is introduced and expressed in zebrafish. First, the 414 bp human cyt $b_5$ gene was amplified from RNA extracts of HeLa cells using RT-PCR, and the amplicon was subsequently sequenced to confirm that it was intact. Next, cyt $b_5$ was cloned into the pEGFP-N3 vector, which also encodes a fluorescent gene. One-cell stage zebrafish embryos were microinjected with the recombinant vector containing the cyt $b_5$ gene. Fluorescence microscopy confirmed high expression of the fluorescent gene in the injected fry compared to the non-fluorescent control fry. Finally, we extracted RNA from the injected fry and performed RT-PCR to determine whether the human cyt $b_5$ gene is expressed in the transgenic zebrafish. Sequencing analysis further confirmed that the cloned human cyt $b_5$ gene was intact. The transgenic zebrafish model produced in this study will be a useful tool to study therapeutic approaches to cure various diseases related to the deficiency of functional human cyt $b_5$ as well as tools for cloning useful genes in fish.