• The Plant Pathology Journal
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• v.38 no.6
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• pp.616-628
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• 2022

Resistance to pyraclostrobin due to a single nucleotide polymorphism at 143rd amino acid position on the cytochrome b gene has been a major source of concern in red pepper field infected by anthracnose in Korea. Therefore, this study investigated the response of 24 isolates of C. acutatum and C. gloeosporioides isolated from anthracnose infected red pepper fruits using agar dilution method and other molecular techniques such as cytochrome b gene sequencing, polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), and allele-specific polymerase chain reaction (PCR). The result showed that four isolates were resistant to pyraclostrobin on agar dilution method and possessed GCT (alanine) codon at 143rd amino acid position, whereas the sensitive isolates possessed GGT (glycine). Furthermore, this study illustrated the difference in the cytochrome b gene structure of C. acutatum and C. gloeosporioides. The use of cDNA in this study suggested that the primer Cacytb-P2 can amplify the cytochrome b gene of both C. acutatum and C. gloeosporioides despite the presence of various introns in the cytochrome b gene structure of C. gloeosporioides. The use of allele-specific PCR and PCR-RFLP provided clear difference between the resistant and sensitive isolates. The application of molecular technique in the evaluation of the resistance status of anthracnose pathogen in red pepper provided rapid, reliable, and accurate results that can be helpful in the early adoption of fungicide-resistant management strategies for the strobilurins in the field.

• Journal of Microbiology
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• v.37 no.3
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• pp.154-158
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• 1999

The arginine residue at position 243 (Arg 243) of the yeast transcription factor, Gcn4p, is invariably conserved among bZIP transcription factors. Using site-directed oligonucleotide saturation mutagenesis involving two-step polymerase chain reaction (PCR) amplification, random mutations were successfully introduced at the codon of 243 in the basic domain of Gcn4p. This mutant library was transformed ito Gcn4p defective yeast strain and selected for the transcriptionally active colonies. All colonies which were transcriptionally active had arginines in the codon 243. In this study, the strand preference by Taq polymerase during mutagenesis was also tested. Oligonucleotides were specially designed to test whether or not the polymerase was preferred using the strand as a template. A population of randomly mutated products were cloned into an appropriate vector and characterized by DNA sequencing analysis. Saturation mutagenesis which was performed efficiently by this method revealed a strong bias in terms of strand preference of Taq polymerase by an approximate ratio of 3 to 1 in this study.

• Journal of Embryo Transfer
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• v.31 no.1
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• pp.61-64
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• 2016

We developed a polymerase chain reaction (PCR)-based molecular method for sexing and identification using sexual dimorphism between the Zinc Finger-X and -Y (ZFX-ZFY) gene and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) for mitochondrial DNA (mtDNA) cytochrome B (CYTB) gene in meat pieces and commercial sausages from animals of different origins. Sexual dimorphism based on the presence or absence of SINE-like sequence between ZFX and ZFY genes showed distinguishable band patterns between male and female DNA samples and were easily detected by PCR analyses. Male DNA had two PCR products appearing as distinct two bands (ZFX and ZFY), and female DNA had a single band (ZFX). Molecular identification was carried out using PCR-RFLP of CYTB gene, and showed clear species classification results. The results yielded identical information on the sexes and the species of the meat samples collected from providers without any records. The analyses for DNA isolated from commercial sausage showed that pig was the major source but several sausages originated from chicken and Atlantic cod. Applying this PCR-based molecular method was useful and yielded clear sex information and identified the species of various tissue samples originating from livestock.

• Journal of Oral Medicine and Pain
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• v.21 no.2
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• pp.243-253
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• 1996

In order to be utilized as a database in forensic identification and parentage test, allelic frequency and genotype distribution of short tandem repeat(STR) F13B locus was analysed by polymerase chain reaction in 210 Korean adults who are not related. The results were as follows. 1. 3 alleles and 56 genotypes of F13B locus were detected and heterozygosity value was 48.6% and allelic diversity value was 0.639 and the power of discrimination was 0.804. 2. The observed each alleles and allelic frequency was 8(0.069), 9(0.193), 10(0.738). In conclusion, the allelic frequency of STR F13B locus in the Korean is considered as an useful DNA allelic profile for forensic identification, but it should be used with several other STR locus to get definitive conclusion of analysis for individual identification and parentage testing.

• Korean Journal of Veterinary Research
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• v.39 no.6
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• pp.1151-1160
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• 1999

This study was conducted to detect the toxoplasma-specific DNA in peripheral blood collected from cats experimentally infected with Toxoplasma gondii (RH strain) and from domiciled cats by B1 gene-base polymerise chain reaction(PCR). The sensitivity of oligonucleotide primer, T-1 & T-2, designed from toxoplasma B1 gene amplification method was compared with parasite detection by mouse inoculation(MI). And also, latex agglutination test(LAT) and indirect fluorescent antibody test(IFAT) were conducted to detect the fluctuation of serum antibodies compared with the detection of toxoplasma by PCR and MI. Toxoplasma B1 gene PCR was shown consistently high sensitivity and the results obtained by PCR agreed completely with those from MI. All blood samples collected before infection with T gondii gave negative results by PCR and MI. Also, toxoplasma Bl gene PCR was not cross reaction with Neospora caninum DNA and normal cat leucocyte as controls. The toxoplasma-specific DNA was detected by PCR in blood of 5 cats experimentally infected with T gondii 6 days after infection and the detection of this specific-DNA was long lasted in blood for 64 days after infection. The detection of toxoplasma-specific DNA by PCR could be identified as few as 10 tachyzoites and the isolation of T gondii by MI could be isolated as few as 1 tachyzoite from tenfold serial dilution of T gondii with normal cat blood, respectively. In healthy domiciled cats, the toxoplasma-specific DNA and the parasite were detected and isolated in blood from 3 of 56(5.3%) cats by both PCR and MI, respectively. In the results of antibody test from the total 56 heads of healthy domiciled cats, the positive rates are 15(26.7%) by LAT and 19(33.9%) by IFAT. These results suggest that PCR detection of toxoplasma can be applied as a sensitive and specific diagnostic and research tool.

• Journal of the Korean Chemical Society
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• v.48 no.5
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• pp.483-490
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• 2004

We have developed a novel polymerase chain reaction (PCR)-microchip gel electrophoresis (MGE) method based on the sieving gel mixture of commercially available poly(vinylpyrrolidone) (PVP) and hydroxy ethyl cellulose (HEC) for the rapid detection and diagnosis of the orf virus (ORFV) from Korean indigenous goat. After amplification of 594-bp DNA fragment from the B2L gene of ORF virus, the amplicon was analyzed by the MGE separation. The glass microfluidic chip (64 mm total length (36 mm effective length)${\times}$90 ${\mu}$m width${\times}$20 ${\mu}$m depth) allowed the fast detection and diagnosis of ORFV in the mixture of 1.0% PVP ($M_r$ 360,000) and 1.0% HEC ($M_r$250,000) as a sieving matrix with better resolution and reproducibility of DNA fragments. Under the electric field of 277.8 V/cm, the 594-bp DNA was analyzed within 4 min. Compared to traditional slab gel electrophoresis, the PCR-MGE method was twenty times faster and an effective clinical method for the quantitative analysis of ORFV.

• The Plant Pathology Journal
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• v.39 no.5
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• pp.522-527
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• 2023

The occurrence of postharvest kiwifruit rot has caused great economic losses in major kiwifruit-producing countries. Several pathogens are involved in kiwifruit rot, notably Botryosphaeria dothidea, and Diaporthe species. In this study, a recombinase polymerase amplification (RPA) assay was developed for the rapid and sensitive detection of the pathogens responsible for posing significant threats to the kiwifruit industries. The RPA primer pairs tested in this study were highly specific for detection of B. dothidea and D. eres. The detection limits of our RPA assays were approximately two picograms of fungal genomic DNA. The optimal conditions for the RPA assays were determined to be at a temperature of 39℃ maintained for a minimum duration of 5 min. We were able to detect the pathogens from kiwifruit samples inoculated with a very small number of conidia. The RPA assays enabled specific, sensitive, and rapid detection of B. dothidea and D. eres, the primary pathogens responsible for kiwifruit rots in South Korea.

• Clinical and Experimental Pediatrics
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• v.49 no.9
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• pp.983-986
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• 2006

Purpose : The purpose of this study was to investigate the presence of Bartonella henselae DNA, which is known as an etiologic agent of lymphadenitis, in fleas from dogs. Methods : The Bartonella henselae infection was investigated in 42 fleas from 22 dogs in Korea. By using seminested PCR targeting pap31 gene, B. henselae DNA was amplified from fleas. Results : B. henselae DNA was detected in seven fleas (7 of 42 fleas, 16.7 percent) from four dogs (4 of 22 dogs, 18.2 percent). To confirm these findings, we performed sequencing and identified the seven PCR products. Sequence analysis revealed that six sequences belonged to Huston-1 genogroup and one sequence to Marseille genogroup. Conclusion : These results may suggest that dogs could be an important source of B. henselae infection in children in Korea. This is the first report about the detection of B. henselae in fleas from dogs in Korea.

• Childhood Kidney Diseases
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• v.12 no.1
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• pp.11-22
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• 2008

Telomeres consist of tandem guanine-thymine(G-T) repeats in most eukaryotic chromosomes. Human telomeres are predominantly linear, double stranded DNA as they ended in 30-200 nucleotides(bases,b) 3'-overhangs. In DNA replication, removal of the terminal RNA primer from the lagging strand results in a 3'-overhang of uncopied DNA. This is because of bidirectional DNA replication and specificity of unidirectional DNA polymerase. After the replication, parental and daughter DNA strands have unequal lengths due to a combination of the end-replication problem and end-processing events. The gradual chromosome shortening is observed in most somatic cells and eventually leads to cellular senescence. Telomere shortening could be a molecular clock that signals the replicative senescence. The shortening of telomeric ends of human chromosomes, leading to sudden growth arrest, triggers DNA instability as biological switches. In addition, telomere dysfunction may cause chronic allograft nephropathy or kidney cancers. The renal cell carcinoma(RCC) in women may be less aggressive and have less genomic instability than in man. Younger patients with telomere dysfunction are at a higher risk for RCC than older patients. Thus, telomeres maintain the integrity of the genome and are involved in cellular aging and cancer. By studying the telomeric DNA, we may characterize the genetic determinants in diseases and discover the tools in molecular medicine.

• Asian-Australasian Journal of Animal Sciences
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• v.32 no.1
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• pp.23-30
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• 2019

Objective: Recent studies have demonstrated that lin-28 homolog B (LIN28B)/miRNA let-7 (let-7) plays a role in the regulation of pubertal onset in mammals. However, the role of LIN28B/let-7 in the onset of ovine puberty remains unknown. We cloned the Duolang sheep Lin28B cDNA sequence, detected the expression change of LIN28B, let-7a and let-7g in hypothalamus, pituitary and ovary tissues at three different pubertal stages. Methods: The reverse transcriptase polymerase chain reaction (RT-PCR) was used to clone the cDNA sequence of LIN28B gene from Duolang sheep and the bioinformatics methods were applied to analyze the amino acid sequence of LIN28B protein. The mRNA expression levels of the LIN28B gene at different pubertal stages were examined by real time RT-PCR. Results: LIN28B cDNA of Duolang sheep was cloned, and two transcripts were obtained. The amino acid sequence of transcript 1 shares 99.60%, 98.78%, and 94.80% identity with those of goat, wild yak and pig, respectively. Strong LIN28B mRNA expression was detected in the hypothalamus, pituitary, ovary, oviduct and uterus, while moderate expression was found in the liver, kidney, spleen and heart, weak expression was observed in the heart. No expression was found in the lungs. Quantitative real-time PCR (QPCR) and western-blot analysis revealed that the LIN28B was highly expressed in the hypothalamus and ovary at prepuberty stages, and this expression significantly decreased from the prepuberty to puberty stages (p<0.05). Markedly increased levels of mRNA expression were detected in the pituitary from prepuberty to puberty (p<0.05) and then significantly decreased from puberty to post-puberty (p<0.05). The expression levels of let-7a and let-7g showed no significant changes among different pubertal stages (p>0.05). Conclusion: These results provided a foundation for determining the functions of LIN28B/let-7 and their role in the onset of sheep puberty.