• Korean Journal of Microbiology
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• v.42 no.1
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• pp.19-25
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• 2006

This study was undertaken to develop PCR primers for the identification and detection of Streptococcus mutans (by)using species-specific forward and universal reverse primers. These primers targeted the variable regions of the 16S ribosomal RNA coding gene (rDNA). The primer specificity was tested against 11S. mutans strains and 10 different species (22 strains) of oral bacteria. The primer sensitivity was determined by testing serial dilutions of the purified genomic DNA of S. mutans ATCC $25175^T$. The data showed that species-specific amplicons were obtained from all the S. mutans strains tested, which was not observed in the other species. The direct and nested PCR could detect as little as 2 pg and 2 fg of the chromosomal DNA from S. mutans ATCC $25175^T$, respectively. This shows that the PCR primers are highly sensitive and applicable to the detection and identification of S. mutans.

• Journal of Genetic Medicine
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• v.10 no.1
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• pp.33-37
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• 2013

Purpose: Myotonic dystrophy 1 (DM1, OMIM 160900) is an autosomal-dominant muscular disorder caused by an expansion of CTG repeats in the 3' UTR of the DMPK gene. Variable expansions of CTG repeats preclude the accurate determination of repeat size. We tried to show the clinical and analytical validity of the application of Southern blotting after long-range PCR was demonstrated in Korean DM1 patients. Materials and Methods: The Southern blotting of long-range PCR was applied to 1,231 cases with clinical suspicion of DM1, between 2000 and 2011. PCR was performed using genomic DNA with forward 5'-CAGTTCACAACCGCTCCGAGC-3' and reverse 5'-CGTGGAGGATGGAACACGGAC-3' primers. Subsequently, the PCR fragments were subjected to gel electrophoresis, capillary transfer to a nylon membrane, hybridization with a labeled (CAG)10 probe. The correlation between clinical manifestations and the CTG repeat expansions were analyzed. Results: Among a total of 1,231 tested cases, 642 individuals were diagnosed with DM1 and the range of the detected expansion was 50 to 2,500 repeats; fourteen cases with mild DM1 ($75{\pm}14$ repeats), 602 cases with classical DM1 ($314{\pm}143$ repeats), and 26 cases with congenital DM1 ($1,219{\pm}402$ repeats). The positive and negative predictive values were 100%. The age at test requested and the CTG repeat numbers were inversely correlated (R=-0.444, P<0.01). Conclusion: This study indicates that Southern blotting after long-range PCR is a reliable diagnostic method DM1.

• Asian-Australasian Journal of Animal Sciences
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• v.20 no.3
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• pp.316-327
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• 2007

In this study, a Korean native cattle strain (Hanwoo) evidencing high performance in terms of both meat quality and quantity was employed in the generation of 150,000 BAC clones with an average insert size of 140 kb, and corresponding to about a 6X coverage of bovine chromosomal DNA. The BAC clones were pooled in a mini-scale via three rounds of a pooling protocol, and the efficiency of this pooling protocol was evaluated by testing the accuracy of accessibility to the positive clones, via a PCR-based screening method. Two sets of primers designed from each of two known genes were tested, and each yielded 2 or 3 positive clones for each gene, thereby indicating that the BAC library pooling system was appropriate with regard to the accession of the target BAC clones. Analyses of $3.3{\times}10^6$ base pairs obtained from the 7,090 BAC end sequence (BES) showed that 34.88% of the DNA sequence harbored the repetition sequence. Analysis of the 7,090 BES to the $1^{st}$ and $2^{nd}$ generation radiation hybrid map of the cattle genome, using the COMPASS program designed for the construction of a cattle-human comparative mapping, resulted in the localization of a total of 1,374 clones proximal to 339 $1^{st}$ generation markers, and 1,721 clones proximal to 664 $2^{nd}$ generation markers. Collectively, the BAC library and pooling system of the BAC clones from the Korean cattle, coupled with the chromosome-localized BAC clones, will provide us with novel tools for the excavation of desired clones for genome mapping and sequencing, and will also furnish us with additional information regarding breed differences in cattle.

• Journal of Microbiology and Biotechnology
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• v.27 no.11
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• pp.2044-2051
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• 2017

The main pathological hallmark of Alzheimer's disease is the deposition of amyloid-beta ($A{\beta}$) peptides in the brain. $A{\beta}$ has been widely used to mimic several aspects of Alzheimer's disease. However, several characteristics of amyloid-induced Alzheimer's disease pathology are not well established, especially in mice. The present study aimed to develop a new Alzheimer's disease model by investigating how $A{\beta}$ can be effectively aggregated using prokaryotes and eukaryotes. To express the $A{\beta}42$ complex in HEK293 cells, we cloned the $A{\beta}42$ region in a tandem repeat and incorporated the resulting construct into a eukaryotic expression vector. Following transfection into HEK293 cells via lipofection, cell viability assay and western blotting analysis revealed that exogenous $A{\beta}42$ can induce cell death and apoptosis. In addition, recombinant His-tagged $A{\beta}42$ was successfully expressed in Escherichia coli BL21 (DE3) and not only readily formed $A{\beta}$ complexes, but also inhibited the proliferation of SH-SY5Y cells and E. coli. For in vivo testing, recombinant His-tagged $A{\beta}42$ solution ($3{\mu}g/{\mu}l$ in $1{\times}PBS$ containing $1mM\;Ni^{2+}$) was injected stereotaxically into the left and right lateral ventricles of the brains of C57BL/6J mice (n = 8). Control mice were injected with $1{\times}PBS$ containing $1mM\;Ni^{2+}$ following the same procedure. Ten days after the sample injection, the Morris water maze test confirmed that exogenous $A{\beta}$ caused an increase in memory loss. These findings demonstrated that $Ni^{2+}$ is capable of complexing the 50-kDa amyloid and that intracerebroventricular injection of $A{\beta}42$ can lead to cognitive impairment, thereby providing improved Alzheimer's disease models.

• Korean Journal of Clinical Laboratory Science
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• v.52 no.1
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• pp.18-27
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• 2020

The correct distinction of carbapenem-resistant Enterobacteriaceae (CRE) and ccarbapenemase producing Enterobacteriaceae (CPE) and the rapid detection of CPE are important for instituting the correct treatment and management of clinical infections. Screening protocols are mainly based on cultures of rectal swab specimens on selective media followed by phenotypic tests to confirm a carbapenem-hydrolyzing activity, the rapid carbapenem inactivation method, lateral flow immunoassay, the matrix-assisted laser desorption ionization-time-of-flight test and molecular methods. The CPE is accurate for detection, and is essential for the clinical treatment and prevention of infections. A variety of phenotypic methods and gene-based methods are available for the rapid detection of carbapenemases, and these are expected to be routinely used in clinical microbiology laboratories. Therefore, to control the spread of carbapenemase, many laboratories around the world will need to use reliable, fast, high efficiency, simple and low cost methods. Optimal effects in patient applications would require rapid testing of CRE to provide reproducible support for antimicrobial management interventions or the treatment by various types of clinicians. For the optimal test method, it is necessary to combine complementary test methods to discriminate between various resistant bacterial species and to discover the genetic diversity of various types of carbapenemase for arriving at the best infection control strategy.

• Korean Journal of Veterinary Service
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• v.34 no.4
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• pp.321-331
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• 2011

Mycobacterium bovis, a member of the M. tuberculosis complex (MTC), is the causative agent of bovine tuberculosis. Detection of M. bovis and M. tuberculosis using conventional culture- and biochemical-based assays is time-consuming. Therefore, a simple and sensitive molecular assay for rapid detection would be of great help in specific situations such as faster diagnosis of bovine tuberculosis (bTB) infection in the abattoirs. We developed a novel multiplex real-time PCR assay which was applied directly to biological samples with evidence of bTB and it was allowed to differentiate between M. bovis and M. tuberculosis. The primers and TaqMan probes were designed to target the IS1081 gene, the multi-copy insertion element in the MTC and the 12.7-kb fragment which presents in M. tuberculosis, not in the M. bovis genome. The assay was optimized and validated by testing 10 species of mycobacteria including M. bovis and M. tuberculosis, and 10 other bacterial species such as Escherichia coli, and cattle lymph nodes (n=113). The tests identified 96.4% (27/28) as M. bovis from the MTC-positive bTB samples using conventional PCR for specific insertion elements IS1081. And MTC-negative bTB samples (n=85) were tested using conventional PCR and the real-time PCR. When comparative analyses were conducted on all bovine samples, using conventional PCR as the gold standard, the relative accuracy of real-time PCR was 99.1%, the relative specificity was 100%, and the agreement quotient (kappa) was 0.976. The detection limits of the real-time PCR assays for M. bovis and M. tuberculosis genomic DNA were 10 fg and 0.1 pg per PCR reaction, respectively. Consequently, this multiplex real-time PCR assay is a useful diagnotic tool for the identification of MTC and differentiation of M. bovis and M. tuberculosis, as well as the epidemiologic surveillance of animals slaughtered in abattoir.

• Interdisciplinary Bio Central
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• v.4 no.3
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• pp.10.1-10.12
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• 2012

Introduction: We investigated frameshift suppressor tRNAs previously reported to use five-base anticodon-codon interactions in order to provide a collection of frameshift suppressor tRNAs to the synthetic biology community and to develop modular frameshift suppressor logic devices for use in synthetic biology applications. Results and Discussion: We adapted eleven previously described frameshift suppressor tRNAs to the BioBrick cloning format, and built three genetic logic circuits to detect frameshift suppression. The three circuits employed three different mechanisms: direct frameshift suppression of reporter gene mutations, frameshift suppression leading to positive feedback via quorum sensing, and enzymatic amplification of frameshift suppression signals. In the course of testing frameshift suppressor logic, we uncovered unexpected behavior in the frameshift suppressor tRNAs. The results led us to posit a four-base binding hypothesis for the frameshift suppressor tRNA interactions with mRNA as an alternative to the published five-base binding model. Conclusion and Prospects: The published five-base anticodon/codon rule explained only 17 of the 58 frameshift suppression experiments we conducted. Our deduced four-base binding rule successfully explained 56 out of our 58 frameshift suppression results. In the process of applying biological knowledge about frameshift suppressor tRNAs to the engineering application of frameshift suppressor logic, we discovered new biological knowledge. This knowledge leads to a redesign of the original engineering application and encourages new ones. Our study reinforces the concept that synthetic biology is often a winding path from science to engineering and back again; scientific investigations spark engineering applications, the implementation of which suggests new scientific investigations.

• Korean Journal of Clinical Laboratory Science
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• v.52 no.4
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• pp.381-388
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• 2020

The quality control of pathological specimens is important for accurate molecular pathology testing. This study evaluated that specimen factors affecting the DNA quality during tissue processing and sample types for BRAF, EGFR, and KRAS mutations tests. One thousand seven hundred and seventy-two molecular pathology tests were investigated for the factors influencing the DNA quality, such as sample type, formalin fixation time, and reexamination status. Cytology samples stored in a saline solution had better DNA quality than commercial cytology preservation. Tissue samples fixed in formalin within 24 hours had better DNA quality than the samples fixed over 24 hours. Between the types of samples, fresh tissue samples and tissue samples with a high tumor cell density had relatively better DNA quality than the formalin-fixed paraffin-embedded (FFPE) tissues and cytology specimens. Of real-time PCR, the non-PNA Ct value increased proportionally with samples held for longer than 24 hours in formalin, and that the formalin-fixed time affects the sample DNA quality. In conclusion, the appropriate tumor cellularity and 10% neutral formalin fixation time are the most important factors for maintaining the DNA quality. These factors should be managed properly for an accurate pathological molecular test to ensure optimal DNA quality.

• The Korea Journal of Herbology
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• v.28 no.4
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• pp.7-16
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• 2013

Objectives : Brassica campestris var narinosa (BCN), Canavalia gladiata DC semen (CGD) and their combinational prescription (BCN+CGD) have been use to demonstrate to regulate hematopoiesis. In the current study, we investigated whether Brassica campestris var narinosa, Canavalia gladiata DC semen and their combinational prescription is related to hemato-potentiating function using Sca-$1^+$ hematopoietic stem cells (Sca-$1^+HSCs$) as a testing system. Methods : Sca-$1^+HSCs$ isolated from femur in C57bl/6 mice with leukopenia and thrombocytopenia induced by cyclophosphamide (CTX). Then, Real-time PCR was performed to measure the mRNA expression, ELISA and haematopoiesis-related gene (EPO, TPO, IL-3, SCF, c-kit, GM-CSF), the phosphorylation of JAK2, GATA-1 and STAT-5a/b were observed by western blot, and the numbers of $CD117^+/Sca-1^+$ cell and the number of granulocyte erythrocyte monocyte macrophage colony-forming units (CFU-GEMM) and erythroid burst forming units (BFU-E), semisolid clonogenic assay was performed. Result : When Sca-$1^+HSCs$ were treated with Brassica campestris var narinosa, Canavalia gladiata DC semen and their combinational prescription with rIL-3/rSCF, the expression of haematopoiesis-related (EPO, TPO, IL-3, SCF, c-kit, and GM-CSF) were significantly increased at the levels of mRNA as well as production in Sca-$1^+HSCs$. Additionally, CGS enhanced phosphorylation of JAK2, GATA-1, and signal transducer and activator of transcription-5a/b (STAT-5a/b) in Sca-$1^+HSCs$. Furthermore, their combinational prescription (BCN+CGD) significantly enhanced the growth rate of granulocyte erythrocyte monocyte macrophage colony-forming units (CFU-GEMM) and erythroid burst forming units (BFU-E) in vitro. Conclusion : These result suggest that Brassica campestris var narinosa (BCN) and Canavalia gladiata DC have hematopoietic enhancement via hematopoietic cytokine-mediated JAK2/GATA-1/STAT-5a/b pathway, and their combinational prescription (BCN+CGD) has superior hematopoietic enhancement to those of individual extracts.

• Journal of Korean Society of Forest Science
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• v.14 no.1
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• pp.1-20
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• 1972

The trend and achievements of forest genetics research in abroad were investigated through observation tours and reference work and following facts were found to be important aspects which should be adopted in the forest genetics research program in Korea. Because of world wide recognization on the urgency of taking a measure to reserve some areas of the representative forest type on the globe before the extingtion of such forest type as the results of continuous exploitations of the natural forests to meet the timber demand all over the world, it is urgently needed to take a measure to reserve certain areas of natural stand of Pinus koraiensis, Pinus parviflora, Pinus densiflora f. erectra, Abies koreana, Quercus sp., Populus sp., etc. as gene pool to be used for the future program of forest tree improvement. And the genetic studies of those natural forest of economic tree species are also to be performed. 1. Increase of the number of selected tree for breeding purpose. Because of the fact that the number of plus tree at present is too small to carry out selection program for tree improvement, particularly for the formation of source population for recurrent selection of parent trees of the 2nd generation seed orchard it is to be strongly emphasized to increase the number of plus tree by alleviating selection criteria in order to enlarge the population size of plus trees to make the selection program more efficient. 2. Progeny testing More stress should be placed on carrying out progeny testing of selected trees with open pollinated seeds. And particular efforts are to be made for conducting studies on adult/juvenile correlation of important traits with a view to enable to predict adult performances with some traits revealed in juvenile age thus to save time for progeny testing. 3. Genotype-environment interaction Studies on genotype and environment interaction should be conducted in order to elucidate whether the plus trees selected on the good site express their superiority on the poor site or not and how the environment affect the genotype. And the justification of present classification of seed distribution area should be examined. 4. Seed orchard of broad leaf tree species. Due to the difficulty of accurate comparison of growth rate of neighbouring trees of broad leaf tree species in natural stand, it is recommended that for the improvement of broad leaf trees a seedling seed orchard is to be made by roguing the progeny test plantation planted densely with control pollinated seedlings of selected trees. 5. Breeding for insect resistant varieties. In the light of the fact that the resistant characteristics against insect such as pine gall midge (Thiecodiplosis japonensis U. et I.) and pine bark beetle (Myelophilus pinipera L.) are highly correlated with the amount and quality of resin which are known as gene controlled characteristics, breeding for insect resistance should be carried out. 6. Breeding for timber properties. With the tree species for pulp wood in particular, emphasis should be placed upon breeding for high specific gravity of timber. 7. Introduction of Cryptomeria and Japanese Cypress In the light of the fact that the major clones of Cryptomeria are originated from Yoshino source and are being planted up to considerably north and high elevation in Japan, those species should be examined on their cold resistance in Korea by planting them in further northern part of the country.