• 한국축산식품학회지
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• 제27권2호
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• pp.209-215
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• 2007

본 연구는 mt DNA 12S rRNA 유전자의 PCR-RFLP 분석기법을 이용하여 다양한 식육자원 및 각종 가공 육제품의 원료육에 대한 정확하고 재현성 높은 축종 및 육종 감별기술을 개발하기 위하여 수행되었다. 국내에서 유통되고 있는 9종류 축종(소, 돼지, 양, 염소, 말, 사슴, 닭, 오리 및 칠면조)의 육류로부터 12S rRNA유전자의 특정 염기서열을 포함하는 primer를 설계 제작하여 PCR-RFLP 분석을 실시하였다. 각 공시축의 근육조직으로부터 genomic DNA를 추출하고 PCR 증폭 반응을 수행한 후 얻어진 PCR 증폭산물(약 455 bp)을 Tsp5091와 MboI 제한효소로 각각 절단한 결과 Tsp5091 제한효소는 포유류 6종간에서 그리고 MboI 제한효소는 가금류 3종간에서 명확한 차이를 보이는 종 특이적인 PCR-RFLP profile을 검출하였다. 따라서 본 연구에서 개발한 12S rRNA 유전자의 종 특이적 DNA 분자표지는 각종 원료육 및 가공 육제품의 육종 및 축종 판별에 매우 유용한 동물 종 감별 DNA marker로 이용될 수 있을 것이다.

• Asian-Australasian Journal of Animal Sciences
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• 제17권7호
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• pp.910-913
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• 2004

Insulin-like growth factor binding protein-3 (IGFBP-3) gene is a structural gene associated with the growth and development of the animals. The present investigation was carried out to unravel nucleotide sequence and polymerase chain reactionrestriction fragment polymorphism (PCR-RFLP) of IGFBP-3 gene in buffalo. Genomic DNA was isolated from a total of 157 animals belonging to Murrah, Surti, Jaffarabadi and Nagpuri breeds of Indian riverine buffalo. A 655 bp of IGFBP-3 gene was amplified in all the breeds and amplicons were digested with Hae III, Taq I and Msp I restriction enzymes. On digestion with Hae III yielded single restriction pattern of 8 fragments of sizes 201, 165, 154, 56, 36, 19, 16 and 8 bp in all the animals studied. Similarly Taq I and Msp I also revealed single restriction pattern yielding fragments of sizes 240 and 415 bp and 145 and 510 bp, respectively. This shows nonpolymorphic nature of restriction sites in buffalo. Nucleotide sequencing of 587 bp of IGFBP-3 gene in Murrah buffalo was done and submitted to the GenBank (Accession No. AY304829). Nucleotide sequencing revealed an addition of 4 bases in the intronic region as compared to cattle.

• Restorative Dentistry and Endodontics
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• 제20권2호
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• pp.577-609
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• 1995

Bacteria have been regarded as one of the most important factors in pulpal and periapical diseases. Streptococci are frequently isolated facultative anaerobes in infected root canals. Recently molecular biological techniques have been rapidly progressed. This study was designed to apply the molecular biological tools to the identification and classification of streptococci in the endodontic microbiology. Streptococci isolated from infected root canals were identified with both Vitek Systems and API 20 STREP. Identification results were somewhat different in several strains of streptococci. Eighteen streptococci and enterococcal was difficult so to digest plasmid DNA using Hind III and EcoRI to differentiate strains by restriction enzyme analysis of plasmid DNA. 16S rDNA of chromosome was amplified by polymerase chain reaction(PCR) and then restricition fragment length polymorphism(RFLP) using several restriction enzymes was observed. The molecular mass of 16S rDNA of chromosomal DNA was approximately 1.4kb. There were three to five RFLP patterns using eight restriction enzymes. RFLP patterns digested with CfoI which recognizes four base sequences were identical in all stains. Hind III which recognizes six base sequences could not digest the 16S rDNA. Restriction enzymes which recognize five base sequences were suitable for RFLP pattern analysis. At least three different restriction enzymes were needed to compare each strains. 16S rDNA PCR-RFLP was simple and rapid to differentiate and classify strains and could be used in the epidemiological study of root canal infections.

• 환경위생공학
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• 제14권4호
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• pp.99-109
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• 1999

Enteropathogenic Yersina enterocolitica is an important cause of human and animal disease. Phenotypic and genotypic characteristics currently used to identify Yersinia enterocolitica are not necessarily sufficient to differentiate pathogenic from non-pathogenic strains or to analyze the epidemiology of yersiniae at a molecular level. To improve the characterization of Yersinia enterocolitica, A total of 65 isolates of Yersinia enterocolitica were examined with bioserotyping, antibiotic susceptibilities, PFGE, PCR-ribotyping. Genomic DNA pattern generated by PFGE are highly specific for different strains of an organism and have significant value in epidemiologic investigations. The PFGE analysis of Not I-digested chromosomal DNA of Y. enterocolitica were performed with a CHEF Mapper(Bio-Rad, USA). Not I generated 19 restriction endonuclease digestion profiles(REDP). PCR-ribotyping, performed with primers complementry to conserved regions of 16S and 23S rRNA gene, generated 13 ribotypes. PCR-ribotyping can be considered a good technich for subtyping strains of Y.enterocolitica.

• 한국유기농업학회지
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• 제27권3호
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• pp.353-363
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• 2019

To identify four cyst nematodes (Heterodera schachtii, H. trifolii, H. glycines, H. sojae) that are economically important plant-parasitic nematodes in Korea, restriction fragment length polymorphism (RFLP) by 8 endonucleases (PstI, VspI, AlwI, RsaI, MvaI, EcoRI, Eco72I, Hinf I) was performed based on sequence difference of mitochondrial DNA cytochrome c oxidase subunit I (COI) gene. As a result, species-specific DNA band patterns by RsaI endonuclease were observed in H. schachtii. The specific patterns was in H. trifolii by 3 endonucleases (VspI, AlwI, Hinf I), and was in H. glycines by Hinf I. While, H. sojae was not digested by 4 endonuclease (VspI, AlwI, RsaI, Hinf I). This study showed that four cyst nematodes could be distinguished using RFLP by 4 endonucleases (RsaI, VspI, AlwI, Hinf I) based on the sequence difference of COI gene.

• The Plant Pathology Journal
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• 제35권4호
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• pp.389-392
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• 2019

The studies on detection of the Strawberry mottle virus (SMoV) have been conducted in Poland for breeding programme purpose and for producers of strawberry plant material. Leaf samples collected from infected strawberry plants were grafted on Fragaria sp. Indicators which were maintained in greenhouse for further study. Seven Fragaria vesca var. semperflorens 'Alpine' indicators infected by SMoV were used for the study aimed on molecular characterization of virus isolates. Partial RNA2 was amplified from total nucleic acids using the RT-PCR method. The obtained amplicons separately digested with BfaI, FauI, HaeIII, HincI, and TaqI enzymes showed different restriction profiles. The nucleotide sequences analysis of RNA2 fragment confirmed the genetic diversity of the SMoV isolates as their similarity ranged from 94.7 to 100%. Polish isolates shared 75.7-99.2% identity with sequence of the virus strains from the Czech Republic, the Netherlands, and Canada. Phylogenetic analysis resulted in grouping of the isolates found in Poland together with one of the Czech strain whereas two other from the Czech and the strains from the Netherlands and Canada created the separate cluster.

• Journal of Microbiology
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• 제38권2호
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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.

• 대한수의학회지
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• 제44권4호
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• pp.533-537
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• 2004

In this study, we performed a PCR-RFLP analysis of NADH oxidase gene(nox) for the characterization of porcine intestinal spirochetes isolated from Korea by the comparison with Brachyspira hyodysenteriae and B. pilosicoli reference strains. Eleven strains including four reference strains, B. hyodysenteriae B204, B234, B169, B. pilosicoli P43/6/78 and seven Korean isolates were used. PCR products of 939 bp were amplified using nox-specific primers and digested with two restriction enzymes, Bfm I and Dpn II. In study using Bfm I, both strains showed no difference in fragmented size(197 and 741 bp). When use Dpn II, B. hyodysenteriae showed two bands(209 and 684 bp), however B. pilosicoli showed a single band of 896 bp. Our results indicate that nox-specific PCR-RFLP could be used as a typing method of Brachyspira species and as an epidemiological method for identifying spirochetes isolated from swine.

• Journal of Microbiology
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• 제34권4호
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• pp.295-299
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• 1996

To infer phylogenetic relationships between species of Trichaptum (Polyporaceae), RFLP analyses of PCR-amplified DNAs were accomplished. Regions coding for ITSs of nuclear SSU rRNA genes and for mitochondrial SSU rRNA genes from thirteen strains of four Trichaptum species (T. abietinum, T. biforme, T. fusco-violaceum, and T. laricinum) were amplified and digested with eight restriction enzymes. All the fragmentation patterns were characterized and coded as 0/1 for the absence/presence of fragments. A phylogenetic tree based on the combined data sets was constructed using the Dollo parsimony method. While every two strains of T. abietinum, T. biforme, T. fusco-violaceum, and T. laricinum formed an independent group, the other strains of T. abietimum and T. fusco-violaceum made mixed groupings among compared strains. It is inferred that T. abietinum and T. fusco-violaceum have more variations, possibly geographic or physiological ones, than other species in the genus.

• 한국식물병리학회지
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• 제14권5호
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• pp.519-525
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• 1998

Intraspecific genetic diversity of Korean isolates of Phytophthora drechsleri was investigated based on PCR-RFLP of rDNA along with closely related species in the genus; P. cryptogea, P. melonis, P. erythroseptica, P. cinnamomi, P. cambivora and P. cactorum. Gene regions of nuclear small subunit and internal transcribed spacer (ITS) in rDNA were amplified with polymerase chain reaction and digested with 9 restriction enzymes. Phytophthora species was readily differentiated from each other based on the digestion patterns, however, P. cryptogea was not separable from some isolates of P. drechsleri. Twenty one isolates of P. drechsleri originated from 15 host plants were divided into three distinct groups designated as PdG1, PdG2 and PdG3, respectively. Four isolates in PdG1 were originated from green vegetables and tomato and nine isolates in PdG2 were mainly isolated from medicinal plants. The two groups showed 95.3% homology and four isolates of P. cyptogea came under the groups. However, Eight isolates in PdG3 collected from cucurbits were clearly differentiated from those of PdG1 and PdG2 by 66.5% homology, but completely matched with a Taiwan isolate of P. melonis. Results indicated that three distinct groups exist in Korean isolates of P. drechleri and each group has host preference. In addition, reclassification of the cucurbits isolates are reserved because of their distinct genetic characters from other intraspecific groups in P. drechsleri.