• Journal of Microbiology
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• v.38 no.2
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• pp.66-73
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• 2000

To investigate the genetic relationship among 12 species belonging to the Fusarium section Martiella, Dlaminia, Gibbosum, Arthrosporiella, Liseola and Elegans, the internal transcribed spacer(ITS) regions of ribosomal DNA (rDNA) were amplified with primer pITS1 and pITS4 using the polymerase chain reaction(PCR). After the amplified products were digested with 7 restriction enzymes, restriction fragment length polymorphism (RFLP) patterns were analyzed. The partial nucleotide sequences of the ITS region were determined and compared. Little variation was observed in the size of the amplified product having sizes of 550bp or 570bp. Based on the RFLP analysis, the 12 species studied were divided into 5 RFLP types. In particular, strains belonging to the section Martiella were separated into three RFLP types. Interestingly, the RFLP type of F. solani f. sp. piperis was identical with that of isolates belonging to the section Elegans. In the dendrogram derived from RFLP analysis of the ITS region, the Fusarium spp. examined were divided into two major groups. In general, section Martiella excluding F. solani f. sp. piperis showed relatively low similarity with the other section. The dendrogram based on the sequencing analysis of the ITS2 region also gave the same results as that of the RFLP analysis. As expected, 5.8S, a coding region, was highly conserved, whereas the ITS2 region was more variable and informative. The difference in the ITS2 region between the length of F. solani and its formae speciales excluding F. solani f. sp. piperis and that of other species was caused by the insertion/deletion of nucleotides in positions 143-148 and 179-192.

• Journal of Microbiology
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• v.40 no.4
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• pp.282-288
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• 2002

Since culture of Helicobacter pylori is relatively insensitive and cumbersome, molecular detection and typing of H. pylori isolates are gaining importance for strain differentiation. In the present study genomic DNA of 42 gastric biopsies and H. pylori isolates from corresponding patients were analyzed and compared by PCR-based RFLP assay. The 1,132-bp product representing an internal portion of ureC gene of H. pylori was amplified by PCR and digested with restriction enzymes HindⅢ, AiuⅠ and PvuⅠ. The HindⅢ, AluⅠ and PvuⅠ digestion produced 4, 7, and 2 distinguishable RFLP patterns respectively from 42-H. pylori isolates. By combining all three restriction enzyme digestions, 15 RFLP patterns were observed. However, when PCR products from 42 gastric biopsy specimens were digested by restriction enzymes HindⅢ, AluⅠ and PvuⅠ, we observed 5, 8 and 2 RFLP patterns, respectively. Patterns from 34 of 42 gastric biopsy specimens matched those of corresponding H. pylori isolates from respective patients. Patterns from the remaining eight biopsy specimens differed and appeared to represent infection with two H. pylori strains. The patterns of one strain from each of these biopsies was identical to that of the isolate from corresponding patients and the second pattern presumably represented the co-infecting strain. From the study, it appears that PCR-based RFLP analysis is a useful primary tool to detect and is distinguish H. pylori strains from gastric biopsy specimens and is superior to culture techniques in the diagnosis of infection with multiple strains of H. pylori.

• Asian-Australasian Journal of Animal Sciences
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• v.19 no.4
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• pp.459-462
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• 2006

Prolactin (PRL) plays an important role in promoting mammalian mammary gland development, and milk production during lactation. Therefore the PRL gene was chosen as a candidate gene for milk traits in Holstein dairy cows. PCR-SSCP and PCR-RFLP were used to analyze genetic variations in the 5' regulation region of the PRL gene. In this part of the gene, two new polymorphic sites were detected in the Chinese Holstein dairy cows. One was a XbaI-RFLP locus, and the other was an SSCP locus. Statistical analysis showed that the XbaI-RFLP locus and the SSCP locus had a significant positive effect on milk traits.

• Journal of Life Science
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• v.31 no.1
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• pp.66-72
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• 2021

This study developed a species identification method for the salted opossum shrimp of Acetes japonicus, A. chinensis (Korea, China), A. indicus (I, II), and Palaemon gravieri based on PCR-RFLP markers. Genomic DNA was extracted from the salted opossum shrimp. The COI gene was used to amplify 519 base pairs (bp) using specific primers. The amplified products were digested by Acc I and Hinf I, and the DNA fragments were separated by automated electrophoresis for RFLP analysis. When the amplified DNA product (519 bp) was digested with Acc I, A. japonicus, A. chinensis (Korea), and A. indius (II) showed two fragments, whereas a single band of 519 bp was detected in A. chinensis (China) and A. indius (I). Also, in the RFLP patterns digested by Hinf I, A. chinensis (Korea) and A. chinensis (China) showed a single band of 519 bp, while two fragments were observed in A. japonicus and A. indius (I) and four fragments in A. indius (II). The PCR amplicon of P. gravieri was digested by Acc I into 3 bands of 271, 202, and 46 bp and by Hinf I into a single band of 519 bp. Therefore, salted opossum shrimp-specific RFLP markers showing distinct differences between four species and two sub-species by PCR-RFLP analysis. Thus, the PCR-RFLP markers developed in this study are a good method for identifying the six types of salted opossum shrimp.

• Korean Journal of Microbiology
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• v.36 no.4
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• pp.249-253
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• 2000

The genetic diversity among Korean cyanobacteria was assessed by restriction fragment length polymorphism(RFLP) analysis of PCR products from the phycocyanin locus. Strains of all the genera tested were successfully amplified, and the size of amplified fragments was approximately 700bp. The restriction patterns generated by AluI, MspI, and HaeIII were conserved for strains within each of genera studied and were specific to the genus level. Intrageneric delineation of strains was revealed by the enzyme, CfoI for members of genera Anabeana and Synechocystis. Phenogram derived from the different RFLP patterns revealed a coherent cluster among Anabeana, Chlorogloea, and Synechocystis strains. PC-RFLP methods provided useful tools for classification of cyanobacteria.

• Korean Journal of Veterinary Research
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• v.39 no.1
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• pp.104-110
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• 1999

Field infectious bursal disease viruses (IBDVs) were isolated from IBDV-suspected commercial chickens. The variable region in VP2 gene of six Korean IBDV isolates (K-IBDVs) and IBD vaccines was examined using the reverse transcriptase / polymerase chain reaction-restriction fragment length polymorphism (RT/PCR-RFLP) assay. With all K-IBDVs and vaccine IBDVs, a 474-bp fragment of the VP2 gene was amplified and tested with various restriction enzymes. Restriction enzymes BstNI and StyI differentiated K-IBDV isolates and IBD vaccines into four groups. Restriction enzyme profiles of K-IBDV isolates were different from them of IBD vaccines. K-IBDV isolates except for 310 isolate had specific SspI and TaqI recognition sites, which were recognized in highly virulent IBDVs, but IBD vaccines had no those sites. This study showed that RT/PCR-RFLP assay was thought to be valuable tool for differentiation of IBDVs and identification of highly virulent IBDV.

• Food Science of Animal Resources
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• v.26 no.3
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• pp.375-379
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• 2006

Food labeling regulations require that the meat species in various meat products are accurately declared to the consumer. Substitution or adulteration of costly meat with a cheaper one is one of the most common problems in the meat industry. In this study, PCR-restriction fragment length polymorphism(RFLP) method of the mitochondrial cytochrome b(mt cyt b) gene has been applied for identification of the origin of six mammalian meat species(beef, port horse, goat, mutton and deer) and three poultry meat species(chicken, turkey and duck) as raw materials for meat products. PCR was used to amplify a variable region of mt cyt b gene. Meat species differentiation was determined by digestion of the amplified products with a 359 bp fragment using HaeIII and HinfI restriction enzymes, which generated species-specific RFLP patterns. This PCR-RFLP DNA marker of mt cyt b gene could be very useful for the accurate and reliable identification and discrimination of animal meat species in routine analysis.

• Korean Journal of Clinical Laboratory Science
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• v.47 no.1
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• pp.17-23
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• 2015

A fine needle aspiration biopsy (FNAB) is the primary means of distinguishing benign from malignant in thyroid nodules. However, between 10 and 30% of the FNABs of thyroid nodules are diagnosed as 'indeterminate'. A molecular method is needed to reduce unnecessary surgery in this group. In Korea, most thyroid cancer is classic papillary type and BRAFV600E mutation is highly prevalent. Thus, this study compared the pyrosequencing method with the conventional direct DNA sequencing and PCR-RFLP analysis and investigated the evaluation of preoperative BRAFV600E mutation analysis as an adjunct diagnostic method with routine FNABs. Sixty-five (78.3%) of 83 histopathologically diagnosed malignant nodule revealed positive BRAFV600E mutation on pyrosequencing analysis. In detail, 65 (83.8%) of 78 papillary thyroid carcinomas sample showed positive BRAFV600E mutation. None of 29 benign nodules had in pyrodequencing, direct DNA sequencing and PCR-RFLP. Out of 31 thyroid nodules classified as 'indeterminate' on cytological examination preoperatively, 28 cases turned out to be malignant: 24 papillary thyroid carcinomas. Among that, 16 (66.7%) classic papillary thyroid carcinomas had BRAFV600E mutation. Among 65 papillary thyroid carcinomas with positive BRAFV600E mutation detected by pyrosequencing analysis, each 3 cases and 5 cases did not show BRAFV600E mutation by direct DNA sequencing and PCR-RFLP analysis. Therefore, pyrosequencing was superior to direct DNA sequencing and PCR-RFLP in detecting the BRAFV600E mutation of thyroid nodules (p =0.027). Detecting BRAFV600E mutation by pyrosequencing was more sensitivity, faster than direct DNA sequencing or PCR-RFLP.

• Proceedings of the Microbiological Society of Korea Conference
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• 2002.10a
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• pp.15-21
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• 2002

Terminal-restriction fragment length polymorphism (T-RFLP) analysis has been optimized by using in vitro model community composed of genomic DNAs of known bacterial strains and has been applied to assess the bacterial community structure in marine sediments. The specific fluorescence-labeled terminal restriction fragments (T-RFs) between 39 and 839 base long specifying each strain were precisely measured for known bacterial strains. The addition of a co-solvent (dimethylsulfoxide or glycerol) into PCR reactions has reduced differential PCR amplification. Comparative bacterial community structure was investigated for pristine and polluted sediments. A complex T-RFLP pattern showing complex bacterial community structure was obtained in the pristine sediment, whereas simple T-RFLP pattern (low bacterial diversity) was shown in polluted sediments where caged aquaculture has been conducted for several years. The results of T-RFLP analysis were compared with that of cloning and sequencing 16S rDNA clones from the same sediments. Sequence analysis of 16S rDNA clones (72) of the pristine sediment revealed a diverse collection of lineages, largely of the class Proteobacteria ($6\%$ alpha subdivision, $46\%$ gamma subdivision, $13\%$ delta subdivision, and $3\%$ epsilon subdivision), Nitrospina $(8\%)$, high G+C gram positive $(8\%)$, Verrucomicrobia $(7\%)$, and Planctomycetes $(6\%)$. In the contaminated sediments, 17 $(59\%)$ of the 16S rDNA clones (29) were related to Campylobacter and symbiont of Rimicaris exoculata belonging to epsilon subdivision of Proteobacteria. The results obtained indicated that T-RFLP analysis is a rapid and precise technique for comparative bacterial community analysis.

• Journal of the korean academy of Pediatric Dentistry
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• v.25 no.2
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• pp.292-303
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• 1998

The arbitrary primer polymerase chain reaction(AP-PCR) and Southern blot restriction fragment length polymorphism(RFLP) were used to genotype the cariogenic pathogen S. mutans in children. Following the morphologic chracteristics of colony on selective medium for S. mutans, total genomic DNA from 155 strains was extracted by conventional methods. Among 155 strains, 143 strains (92.3%) were confirmed S. mutans by PCR with dexA gene and 114 strains were used in this study. Three random sequence 10-base oligonucleotide primers were chosen for AP-PCR. The amplified DNA products were separated electrophoretically in a 2% agarose gel containing ethidium bromide and the banding patterns were compared among different strains. For RFLP analysis, DNA was digested with EcoRI and BamHI, separated on a 0.7 % agarose gel and transferred to a nylon membrane. The membrane was probed with a previously characterised 1.6 kilobases (kb) DNA fragment cloned from gtf B gene of S. mutans. The probe was labeled with isotope[$^{32}P-{\alpha}CTP$], and hybridized fragments were detected with intensifying screen. AP-PCR produced 4-8 DNA bands in the 0.25-10 kb regions and distinguished 9, 10 or 12 genotypes, depending on the specific primer used. Southern blot RFLP analysis revealed 2 hybridization patterns consisting of 1 DNA fragments 450, 500 bp. These results indicate that AP-PCR is more discriminative method for genotyping of S. mutans.