• Korean Journal of Agricultural Science
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• v.26 no.2
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• pp.39-48
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• 1999

This study was carried out to investigate the difference and genetic similarity at the level of molecular genetics. Genomic DNA was extracted from blood of Holstein, Korean cattle, Charolais, and hybrid between Korean cattle and charolais and RAPD(random amplified polymorphic DNAs) was analyzed by PCR(polymerase chain reaction). After genetic similarity value from different breeds are analyzed, genetic similarity was estimated by UPGMA(unweighted pair-group method using average). The results obtained from this study can be summarized as follows: 1. When genomic DNA which was extracted from different breeds was subjected to electrophoresis on 1.5% agarose gel, bigger than 12.2kb was appeared. Ratio by absorbance of $A_{260}/A_{280}$ was 1.75~2.10, indicating that genomic DNA was quite pure for RAPD analysis. 2. Different band patterns by RAPD were appeared according to the breeds in cattle. The best primer used to distinguish Holstein from other breeds was 5'-GAC CGC TTG T-3'. 3. A 340bp fragment was amplified in $33.0^{\circ}C$ of annealing temperature for the Holstein and Charolais breeds, but any amplification was not occurred in this annealing temperature for Korean cattle and hybrid. In addition, a 340bp fragment was amplified in $37.5^{\circ}C$ of annealing temperature for the Holstein and Korean cattle, but any amplification was not occurred in this annealing temperature for Charolais and hybrid. For the reaction of PCR. $37.5^{\circ}C$ and $33.0^{\circ}C$ of annealing temperature was shown to be best for genetic marker identification from Holstein, Charolais, and hybrid between Korean cattle and Charolais. 4. When genetic similarity from different breeds are analyzed at the both temperature of $33.0^{\circ}C$ and $37.5^{\circ}C$, the genetic similarity value of Holstein and Korean cattle, Holstein and Charolais, Korean cattle and Charolais, and Korean cattle and hybrid were 0.666~0.777, 0.615~0.666, 0.400~0.461 and 0.857~0.888, respectively. 5. It could be concluded that different breeds are capable of distinguishing by RAPD used random primer 5'-GAC CGC TTG T-3', genetic similarity from different breeds was appeared the higher genetic similarity value of Korean cattle and Charolais than that of Holstein between Korean cattle and Charolais by UPGMA.

• Tuberculosis and Respiratory Diseases
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• v.40 no.2
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• pp.98-103
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• 1993

Background: Inactivation of retinoblastoma gene (Rb) has been observed in a variety of human cancers. Loss of heterozygosity (LOH) of Rb which is a common mode of allelic inactivation of Rb, has been known as a frequent genetic event in small cell lung cancer but it has been detected less frequently in non-small cell lung cancer. To define the role of Rb deletion in lung cancer, we investigated the genomic DNAs of 43 non-small cell lung cancers and 1 small cell lung cancer paired with normal lung tissues obtained by thoracotomy. Methods: The genomic DNAs were obtained by the digestion with proteinase K followed by phenol-chloroform extraction method. The genomic DNAs were digested by restriction endonuclease (EcoRI), separated by agarose gel electrophoresis, transferred to nylon membrane by Southern blot transfer and then hybridized with labelled Rb 1 probe which contains. 1.4 kb sized DNA sequence containing N-terminal portion of Rb. Results: In 26 squamous cell lung cancers, 16 cases were informative after EcoRI digestion and LOH of Rb was found in 10 cases (62.5%). In 17 adenocarcinomas of lung, 11 cases were informative and LOH of Rb was found in five cases (45.4%). The analysis of clinical parameters revealed no significant differences between the two groups with or without LOH of Rb in the aspects of age, sex, degree of differentiation, stage and smoking amount. Conclusions: These results suggest that Rb inactivation is also significantly involved in the molecular pathogenesis of non-small cell lung cancer.

• Korean Journal of Clinical Laboratory Science
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• v.38 no.3
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• pp.196-202
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• 2006

Treatment of hepatitis B virus (HBV) with lamivudine is effective in suppressing virus replication and results in reduced inflammatory activity. However the most troublesome problem of lamivudine treatment is the emergence of lamivudine-resistant strains with amino acid substitution in the YMDD motif of DNA polymerase gene during the treatment. The aim of this study was to determine the mutation of YMDD motif (codon 552) and codon 528 in chronic HBV patients with lamivudine therapy using PCR-direct sequencing and to investigate the relationship between lamivudine mediated HBV mutation and HBeAg. HBV DNA was extracted from serum samples of HBV patients and amplified by nested PCR with two sets of primer pairs selected in HBV DNA polymerase gene. Amplified PCR product was analyzed by 2% agarose gel electrophoresis and direct sequencing. HBV mutation was detected in 124 out of 207 samples (60%). Single mutation was 50.8% for M552I, 43.5% for M552V, 5.7% for M552I/V and the L528M mutation was 67.0%. Double mutation was 43.6% for M552V/L528M, 33.1% for M552I/L528(wild type), 17.7% for M552I/L528M and 5.6% for M552I/V/L528M. Serine mutation at YMDD motif (M552S) was not found and the L528M mutation frequently accompanied M552V type. In this study, the typical difference of frequencies for HBV mutation depending on HBeAg was not found. Moreover, the PCR-direct sequencing method used in this study might be a powerful tool for the mutation study in clinical reference laboratories with high volume.

• Journal of Life Science
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• v.28 no.9
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• pp.1062-1067
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• 2018

Reliable labeling of fish products can reassure consumers regarding the identity and quality of seafoods. Therefore, techniques that can identify adulteration or mislabeling are valuable. To rapidly identify two Trachurus species, Trachurus japonicus and Trachurus novaezelandiae, a highly efficient, rapid, duplex polymerase chain reaction (PCR) having two species-specific primers simultaneously was identified. This species-specific primer focused on a single nucleotide mismatch in the 3'-terminal base of a primer designed in the mitochondrial cytochrome c oxidase (COI) subunit I DNA. To optimize the duplex PCR condition, gradient PCR reactions were conducted to determine the primer annealing temperature and the primer concentration. The PCR's product was observed on the gel, suggesting that DNA molecules may be useful in differentiating the two species. The length of the amplification fragments were 103 bp for Trachurus japonicus and 214 bp for Trachurus novaezelandiae, which, along with the species-specific primer visualized by agarose gel electrophoresis, enabled accurate distinction of the species of the Trachurus genus. These results indicate that the duplex PCR, which has a species-specific primer based on single nucleotide polymorphism (SNP), can be useful for rapidly differentiating the two species of Trachurus. This duplex PCR analysis is simple, rapid, and reliable, and could be beneficial to protecting consumers' rights.

• Journal of Life Science
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• v.26 no.5
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• pp.620-631
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• 2016

Viroids are about 250-400 base pair of short single strand RNA fragments have been associated with economically important plant diseases. Due to the lack of protein expression capacity associated with replication, it is very difficult to diagnosis viroid diseases in serological methods. For detecting viroid at plants, molecular-based techniques such as agarose gel electrophoresis, polyacrylamide gel electrophoresis (PAGE), DNA-hybridization, blotting analysis and conventional RT-PCR are reliable. Real-time RT-PCR methods that grafted on RT-PCR methods with improved confirmation methods have been also utilized. However, they are still labor-intensive, time-consuming, and require personnel with expertise. Loop-mediated Isothermal Amplification (LAMP) method is a nucleic acid amplification method under the isothermal condition. The LAMP methodology has been reported to be simple, rapid, sensitive and field applicable in detecting a variety of pathogens. The results of LAMP method can be colorized by adding a visible material such as SYBR green I, Evagreen, Calcein, Berberine and Hydroxy naphthol blue (HNB) with simple equipment or naked eyes. The combination of LAMP method and nucleic pathogens, viroids, can be used to realize simple diagnosis platform for the genetic point-of care testing system. The aim at this review is to summary viroid-caused diseases and the simple visible approach for diagnosing viroids using Loop-mediated Isothermal Amplification (LAMP) method.

• Polymer(Korea)
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• v.29 no.2
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• pp.135-139
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• 2005

In this study, poly(ethylene imine) (PEI) modified by methoxypoly(ethylene glycol) (mPEG) and folate as a gene carrier was synthesized to decrease cytotoxicity and to improve in vivo targeting. mPEG was modified by glutaric anhydride (GA) to endow carboxylic end group, followed by the activation reaction with EDC (N-ethyl-N'-(3-dimethyl-aminopropyl) carbodiimide) and NHS (N-hydroxysuccinimide). The activated carboxylic end group of mPEG was reacted with the amines of PEI to give mPEG graft PEI. The mPEG-folate-graft-PEI was synthesized by the reaction of mPEG-PEI with folate pre-activated by EDC/NHS. The obtained copolymers were characterized by $^1H-NMR$ and FT-IR. Gel retardation assay and fluorescence measurement indicated that DNA formed the complexes with the synthesized copolymers above N/P charge ratio 2. The size of complexes was ranging from 100 nm to 300 m. In conclusion, we confirmed that the synthesized copolymer have the possibility as a DNA carrier.

• Microbiology and Biotechnology Letters
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• v.31 no.2
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• pp.117-123
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• 2003

For screening of autoregulator receptor gene from Saccharopolyspora erythraea, PCR was performed with primers of receptor gene designed on the basis of amino acid sequences of autoregulator receptor proteins with known function. PCR products were subcloned into the BamHI site of pUC19 and transformed into the E. coli DH5$\alpha$. The isolated plasmid from transformant contained the fragment of 120 bp, which was detected on 2% gel after BamHI treatment. The insert, 120 bp PCR product, was confirmed as the expected internal segment of gene encoding autoregulator receptor protein by sequencing. Southern and colony hybridization using Saccha. erythraea chromosomal DNA were performed with the insert as probe. The plasmid (pEsg) having 3.2 kbp SacI DNA fragment from Saccha. erythraea is obtained. The 3.2 kbp SacI DNA fragment was sequenced by the dye terminator sequencing. The nucleotide sequence data was analyzed with GENETYX-WIN (ver 3.2) computer program and DNA database. frame analyses of the nucleotide sequence revealed a gene encoding autoregulator receptor protein which is a region including KpnI and SalI sites on 3.2 kbp SacI DNA fragment. The autoregulator receptor protein consisting of 205 amino acid was named EsgR by author. In comparison with known autoregulator receptor proteins, homology of EsgR showed above 30%.

• Journal of Microbiology and Biotechnology
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• v.10 no.1
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• pp.27-34
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• 2000

The chloroform and methanol (2;1, v/v) extract from an edible plant, Actinidia arguta Planchon, appeared to possess antitumor activity against human leukemias Jurkat T and U937 cells through inducing apoptosis. The substance in the solvent extract was purified by silica gel column chromatography, preparative TLC, and Sephadex LH-20 column chromatography. Characteristics of the substance analyzed by UV scanning analysis, $^1H$ and $^{13}C$ NMR spectra suggested that the substance belongs to the chlorophyll derivatives-like group. The $IC_{50}$ value of the chlorophyll derivative (Cp-D) determined by MTT assay was $15\mu\textrm{g}/ml$ for Jurkat, $10\mu\textrm{g}/ml$ for U937, and $11.4\mu\textrm{g}/ml$ for HL-60m and was more toxic to these leukemias than to solid tumors or normal fibroblast. In order to elucidate cellular mechanisms underlying the cytotoxicity, the effect of the Cp-D on Jurkat T cells was investigated. When cells were treated with the Cp-D at a concentration of $15\mu\textrm{g}/ml$, [3H]thymidine incorporation declined rapidly and wa undetectable in 1h. However, no significant changes were made in the cell cycle distribution of the cells by 24h. The sub-Gl peak representing apoptotic cells began to be detectable in 36h, at which time apoptotic DNA fragmentation was also detected on agarose gel electrophoresis, demonstrating that the cytotoxic effect of the Cp-D is attributable to the induced apoptosis. Under the same conditions, although the protein level of cyclin-dependent kinases such as cdc4, csk6, cdk2, and cdc2 was not significantly changed until 24h, the kinase activity of all c안 rapidly declined and reached a minimum level within 1-6h and then recovered to the initial level by 12h and sustained until 24h. These results suggest that inactivation of cdks at an inappropriate time during the cell cycle progression in jurkat T cells following a treatment with the Cp-D leads to induction of apoptotic cell death.

• Journal of Periodontal and Implant Science
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• v.35 no.2
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• pp.299-310
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• 2005

The severe form of chronic periodontitis(CP) has been reported to be strongly associated with the presence of allele 2 of composite IL-1B(+3954) and IL-1A(+4845) genetic polymorphisms(genotype positive). However, other studies have reported conflicting findings. These might have resulted from differences in ethnic background and disease entities. The aim of this study was to determine the distribution of IL-1A(+4845), IL-1B(+3954), IL-1B(-511), and IL-1 RN(VNTR) genetic polymorphisms in children as a future Korean population. The study population consisted of 92 children from the Dept. of Pediatric Dentistry, Chonnam National University Hospital. Genomic DNA was obtained from buccal swab. The IL-1A(+4845), IL-1B(+3954), and IL-1B(-511) genes were genotyped by amplifying the polymorphic region using multiplex polymerase chain reaction(PCR), followed by restriction enzyme digestion and gel electrophoresis. IL-1 RN(VNTR) polymorphism were then evaluated by PCR amplification and fragment size analysis in agarose gel. The allele 2 frequency was 41.3%, 4.3%, 47.8%, and 9.9% for IL-1A(+4845), IL-1B(+3954), IL-1B(-511), and IL-1 RN respectively. The frequency of genotype with allele 2 carriage for IL-1A(+4845), IL-1B(+3954), IL-1B(-511), and IL-1 RN was 77.1%, 7.6%, 63.0%, and 15.2% respectively. The allele 2 frequency in IL-1B(+3954) was significantly higher in female than in male population(p<0.05). The negative association was shown between the presence of allele 2 in IL-1B(-511) and in IL-1B(+3954), and the carriage rate of IL-1B(+3954) allele 2 tended to lower in IL-1B(-511) allele 2(P=0.056). Only 7.3% of children carried the composite genotype of IL-1A(+4845) and IL-1B(+3954). These results suggest that the polymorphism of IL-1B(+3954) and the positive composite genotype was relatively rare in Korean population.

• Journal of Life Science
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• v.24 no.9
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• pp.927-934
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• 2014

Sanguinarine, a benzophenanthridine alkaloid originally derived from the root of Sanguinaria canadensis, has been shown to possess antimicrobial, antioxidant, and anti-cancer properties. Although tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is known to induce apoptosis in cancer cells, but not most normal cells and has shown efficacy in a phase 2 clinical trial, development of resistance to TRAIL by tumor cells is a major roadblock. Our previous study indicated that treatment with TRAIL in combination with subtoxic concentrations of sanguinarine sensitized TRAIL-mediated apoptosis in TRAIL-resistant human gastric carcinoma AGS cells; however, the detailed mechanisms are not fully understood. In this study, we show that sanguinarine sensitizes AGS cells to TRAIL-mediated apoptosis as detected by MTT assay, agarose gel electrophoresis, chromatin condensation and flow cytometry analysis. Combined treatment with sanguinarine and TRAIL effectively induced expression of death receptor (DR) 5 but did not affect expression of DR4 and mitogen activated protein kinases signaling molecules. Moreover, the combined treatment with sanguinarine and TRAIL increased the generation of reactive oxygen species (ROS); however, N-acetylcysteine, ROS scavenger, significantly recovered growth inhibition induced by the combined treatment. Taken together, our results indicate that sanguinarine can potentiate TRAIL-mediated apoptosis through upregulation of DR5 expression and ROS generation.