• Korean Journal of Plant Resources
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• v.21 no.3
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• pp.210-215
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• 2008

Gene-expression analysis is increasingly important in biological research, with real-time reverse PCR (RTPCR) becoming the method of choice for high-throughput and accurate expression profiling of selected genes. However, this technique requires important preliminary work for standardizing and optimizing the many parameters involved in the analysis. Plant stress studies are more and more based on gene expression. The analysis of gene expression requires sensitive and reproducible measurements for specific mRNA sequence. Several genes are regulated in response to abitoic stresses, such as salinity, and their gene products function in stress response and tolerance. The design of the primers and TaqMan probes for real-time PCR assays were carried out using the Primer $Express^{TM}$ software 3.0. The PCR efficiency was estimated through the linear regression of the dilution curve. To understand the expression pattern of various genes under salt stressed condition, we have developed a unique public resource of 9 stress-related genes in poplar. In this study, real-time RT-PCR was used to quantify the transcript level of 10 genes (9 stress-related genes and 1 house keeping gene) that could play a role in adaptation of Populus davidiana. Real-time RT-PCR analyses exhibited different expression ratios of related genes. The data obtained showed that determination of mRNA levels could constitute a new approach to study the stress response of P. davidiana after adaptation during growth in salinity condition.

• Asian-Australasian Journal of Animal Sciences
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• v.6 no.4
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• pp.541-547
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• 1993

A phage clone containing the partial ${\alpha}_{S1}$-casein gene was isolated from a bovine genomic library by using mixed probes of ovine ${\alpha}_{S1}$-, ${\beta}$- and ${\kappa}$-casein cDNAs. Restriction enzyme mapping analysis for 14.6 kb revealed that the map was in conflict with the report of Meade et al. (1990), especially in the 3'-end fragment. Sequence analysis of 12.6 kb revealed a high AT/GC ratio (1.64); we have identified eight exon sequences according to the bovine ${\alpha}_{S1}$-casein cDNA sequence. The same exon/intron splice junction sequence was observed between these exons. We suggest that the bovine ${\alpha}_{S1}$-casein gene night contain a minimum of 18 exons and the full length is approximately 18-19 kb.

• Journal of Microbiology and Biotechnology
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• v.19 no.3
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• pp.265-270
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• 2009

An oligonucleotide array was developed to detect and genotype mollicutes based on the internal transcribed spacer (ITS) sequence. The results of the assay were compared with those of a PCR-RFLP assay. The proposed oligonucleotide array containing 5 genus- and 23 species-specific probes was able to detect Mycoplasma species, including M. penetrans and M. spermatophilum, that were not detected by the PCR-RFLP assay. Therefore, the results demonstrated that the proposed oligonucleotide array was effective for the detection and discrimination of 23 species, including an acholeplasma, 21 mycoplasmas, and a ureaplasma, and showed promise as a countermeasure to ensure that biological products are safe and of good quality.

• Fisheries and Aquatic Sciences
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• v.17 no.3
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• pp.339-344
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• 2014

PCR amplification with universal primer is a useful tool for speciation of symbionts in marine eukaryote coupled with robust separation method such as denaturing high performance chromatography (DHPLC). To overcome the biased amplification, clamping PCR is recommended to suppress the amplification of host gene. In this study, we evaluated the efficiency of rare gene detection for two kinds of clamping probes which were successfully utilized for eukaryotic symbiont analysis: C3 linked nucleotide (C3) and peptide nucleic acid (PNA). PNA was 3-4 orders of magnitude higher than that of C3 tested in clamping efficiency and rare gene detection. This represented that PNA could be a more competent clamping probe for the enhancement of PCR amplification for rare symbiont genes.

• 한국균학회소식:학술대회논문집
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• 2014.10a
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• pp.42-42
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• 2014

Mating of tetrapolar mushrooms is regulated by to chromosomal loci, A and B. A locus contains A gene that expresses a homeodomain protein whereas B locus contains multiple pheromones and receptor genes. In order to characterize the mating loci in Korean cultivated strains of Lentinula edodes, one hundred monokaryotic myclelia were isolated from the basidiospores of cultivated strains, including Cham-A-Ram, Sanjo701, and Sanjo707. Both mating loci were amplified using primer sets targeting conserved sequence regions for homeodomain (HD), pheromone, and receptor genes. Subsequent sequence analysis revealed that the Korean strains contained significant variations in the homeodomain of A locus, even within the same A1 or A2 mating type. Similarly, B locus was also highly diversified in the sequences of pheromones and receptors as well as gene organization. These results enabled us to design mating type-specific probes which can distinguish mating type of each strain. The specificity was confirmed by between intra- and inter-strain mating experiment.

• Journal of the Korea Academia-Industrial cooperation Society
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• v.20 no.12
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• pp.117-124
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• 2019

Vibrio cholerae is a very important pathogenic bacterium that has to be monitored in seafood and ships' ballast water. Various methods have been developed to identify this bacterium, yet these methods are time-consuming and have limitations for their sensitivity to detect contamination. The purpose of the present study was to develop a robust and reliable method for identifying V. cholerae. Peptide nucleic acid (PNA) probes were developed to use for PNA-based asymmetrical real-time PCR techniques. The toxigenic Cholera enterotoxin subunit B (ctxB) gene was selected as a target for detecting V. cholerae and the gene was synthesized as a positive template for conventional and real-time PCR. Real-time PCR primers and PNA probes were designed and standard curves were produced for the quantitative analysis. The selected PNA probes reacted specifically to V. cholerae without any ambiguity, even among closely related species, and the detection limit was 0.1 cfu/100 mL. Taken together, the PNA probes and asymmetrical qPCR methods developed in this present study could contribute to the rapid, accurate monitoring of V. cholerae in marine environments, and as well as in seafood and ships' ballast waters.

• Proceedings of the Korean Society for Applied Microbiology Conference
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• 1986.12a
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• pp.529.2-529
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• 1986

pSp-Si (1.6kbp) was originally found in pediococcus halophilus to be a cryptic multicopy-plasmid. Hoping that the plasmid can also replicate in Bacillus subtilis, protoplast transformation of strain 207-25 (recE) was performed using pSP-Sl onto which was added the marker of tmrB8 (on 4.9 kbp EcoRI fragment ) or tmrB+ (on 0.9 kbp xbaI fragment) gene. Though the tmrB8 gene can expres tunicamycin-resistance at the single copy state, and the tmrB+ gene exerts the resistance only at the multicopy state, we could not confirm the replication of pSP-Sl (tmrB8) or pSP-Sl(tmrB+) in B. subtilis. During the experiment, however, we unexpectedly found that the circularized 0.9 kbp xgaI fragment (tmrB+) itself, which had no replication origin, could transform strain 207-25 to tunicamycin-resistant by protoplast transformation. Southern hybridization analyses with tmrB+ and other probes revealed the integration of the fragment at a single copy state into a position other than the homologous tmrB gene. This recE independent integration of another tmrB+ gene into the chromosome may contribute to the tunicamycinresistance in the transformants.

• BMB Reports
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• v.36 no.4
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• pp.349-353
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• 2003

To explore the application of DNA chip technology for the detection and typing of Human Papillomavirus (HPV), the HPV6, 11, 16 and 18 gene fragments were isolated and printed onto aminosilane-coated glass slides by a PixSys 5500 microarrayer as probes to prepare the HPV gene chips. HPV samples, after being labeled with fluorescent dye by restriction display PCR (RD-PCR) technology, were hybridized with the microarray, which was followed by scanning and analysis. The experimental condition for preparing the HPV gene chips was investigated, and the possibility of HPV genotyping using gene chips was discussed. The technique that was established in this study for preparing HPV gene chips is practical. The results of the present study demonstrated the versatility and inspiring prospect of using this technology to detect and genotype HPV.

• Journal of Plant Biotechnology
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• v.34 no.1
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• pp.75-80
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• 2007

The release of genetically modified organisms ($GMO_{s}$) into the environment has the potential risks regarding the possibility of gene transfer from $GMO_{s}$ to natural organisms and this needs to be evaluated. This study was conducted to monitor the possible horizontal gene transfer from herbicide-resistant zoysiagrass (Zoysia japonica Steud.) to indigenous microorganisms. We have first examined the effect of field-released GM zoysiagrass on the microbial flora in the gut of locust (Locusts mlgratoria). The microbial flora was analyzed through determining the 165 rDHA sequences of microorganisms. The comparison of the microbial flora in the gut of locusts that were captured at the field of GM zoysiagrass and of wild-type revealed that there is no noticeable difference between these two groups. This result indicates that the GM zoysiagrass does not have negative impact on microbial flora in the gut of locust. We then investigated whether the horizontal gene transfer occurred from GM zoysiagrass to microbes in soil, rhizosphere and faecal pellets from locusts by utilizing molecular tools such as Southern hybridization and polymerase chain reaction (PCR). When the total DNAs isolated from microbes in GM zoysiagrass and in wild-type zoysiagrass fields were hybridized with probes for bar or hpt gene, no hybridization signal was detected from both field isolates, while the probes were hybridized with DNA from the positive control. Absence of these genes in the FNAs of soil microorganisms as well as microbes in the gut of locust was further confirmed by PCR. Taken together, our data showed that horizontal gene transfer did not occur in this system. These results further indicate that frequencies of transfer of engineered plant DNA to bacteria are likely to be negligible.

• Proceedings of the Korea Society of Environmental Biology Conference
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• 2001.12a
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• pp.93-93
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• 2001