• 한국약용작물학회지
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• 제18권6호
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• pp.379-388
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• 2010

Adenophora racemosa is recently reported as a new Korean endemic plant species. However, the phylogenetic relationship of this genus has been controversial due to the morphological similarity and frequent morphological change of aerial parts. To verify the phylogenetic position of Adenophora racemosa and phylogenetic relationship of genus Adenophora, we analyzed the internal transcribed spacer (ITS) sequence of nuclear ribosomal DNA (nrDNA) and random amplified polymorphic DNA (RAPD) using 21 individual of 6 Adenophora species, A. verticillata, A. divaricata, A. racemosa, A. remotiflora, A. stricata and A. tetraphylla. In comparative analysis of the nrDNA-ITS sequences, we could not found not only any species specific nucleotide sequence but also could not estimated their inter or intra species. In the phylogenic analysis based on the RAPD derived DNA polymorphism, Adenophora species were classified into four groups by clustering analysis of the UPGMA. These results suggest that the DNA fingerprinting based on RAPD is more suitable than nrDNA-ITS sequence for the phylogenetic analysis of Adenophora species.

• Plant Resources
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• 제3권3호
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• pp.211-218
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• 2000

Intraspecific genetic relationship of 19 variation types of the Yam (Dioscorea alata) classified by their external morphological characteristics such as leaf and tuber shape were assessed by DNA using random and specific primer. Twenty two out of 113 primers (100 random[10-mer] primers, two 15 mer [M13 core sequence, and (GGAT)$_4$ sequence]) had been used in PCR-amplification. Only 12 primers, however, were success in DNA amplification in all of the analyzed plants, resulting in 93 randomly and specifically amplified DNA fragments. The analyzed taxa showed very high polymorphisms(69 bands, 71.0 %), allowing individual taxon to be identified based on DNA fingerprinting. Monomorphic bands among total amplified DNA bands of each primer was low under the 50%. Similarity indices between accessions were computed from PCR(polymerase chain reaction) data, and genetic relationships among intraspecific variations were closely related at the levels ranging from 0.66 to 0.90. These DNA data were not matched well with those of morphological characters since they were divided into two major groups at the similarity coefficient value of 0.70. Therefore, Grouping of species into variation types by mainly morphological charactistics was suggested unreasonable.

• Journal of Microbiology and Biotechnology
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• 제21권12호
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• pp.1336-1344
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• 2011

This study details and examines a novel ligation-mediated polymerase chain reaction (LM-PCR) method. Named the LM-PCR/Shifter, it relies on the use of a Class IIS restriction enzyme giving restriction fragments with different 4-base, 5' overhangs, this being the Shifter, and the ligation of appropriate oligonucleotide adapters. A sequence of 4-base, 5' overhangs of the adapter and a 4-base sequence of the 3' end of the primer(s) determine a subset of the genomic restriction fragments, which are amplified by PCR. The method permits the differentiation of bacterial species strains on the basis of the different DNA band patterns obtained after electrophoresis in polyacrylamide gels stained with ethidium bromide and visualized in UV light. The usefulness of the LM-PCR/Shifter method for genotyping is analyzed by a comparison with the restriction endonuclease analysis of chromosomal DNA by the pulsed-field gel electrophoresis (REA-PFGE) and PCR melting profile (PCR MP) methods for isolates of clinical origin. The clustering of the LM-PCR/Shifter fingerprinting data matched those of the REA-PFGE and PCR MP methods. We found that the LM-PCR/Shifter is rapid, and offers good discriminatory power and excellent reproducibility, making it a method that may be effectively applied in epidemiological studies.

• 한국한의학연구원논문집
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• 제1권1호
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• pp.471-476
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• 1995

Natural products used for oriental medicine often come from various geographical sources, after several different distribution channels. Therefore some form of quality control procedure is required to safeguard naturl products for prescriptions purposes. To achieve this, systematic apprroaches such as morphological examination, microscopic analysis of powdered herbs and chemical analysis can be carried out. However, to ensure absolute criteria for quality assurance of natural products, DNA fingerprinting method such as RAPD(Random amplified polymorphism DNA) analysis can be used for authentication of natural products for authenticatin of natural products. In this study, warious oligonucleotide primers will be synthesized for the detection of RAPD markers and also parameters of affecting PCR(Polymerase Chain Reaction) in the detection of RAPD markers of rare and high-priced natural products will be studied with genomic DNA of chosen samples.

• 대한수의학회지
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• 제45권2호
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• pp.199-205
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• 2005

DNA fingerprint patterns of 8 Brucella reference strains and 15 B. abortus field isolates were characterized by repetitive element sequence-based PCR (rep-PCR) using BOX- and ERIC-primers in this study. AMOS PCR differentiated all Brucella field isolates from B. abortus RB51, a vaccine strain by producing a B. abortus-specific 498 bp band. Rep-PCR using BOX-primer produced 13 to 18 bands with sizes of between 230 and 3,300 bp, and discriminated Brucella strains to the species level except B. canis and B. suis. PCR products amplified with ERIC primers were, however, not appropriate for differentiating the Brucella isolates. DNA fingerprint patterns for all B. abortus field isolates were identical among them and were put on one cluster with B. abortus biovar 1 reference strain in the dendrogram, indicating they were highly clonal. These results suggested that rep-PCR using BOX primer might to be a useful tool for calculating genetic relatedness among the Brucella species and for the study of brucellosis epidemiology.

• 미생물학회지
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• 제48권4호
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• pp.275-283
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• 2012

해양 해면 Asteropus simplex를 제주도에서 채집하여 배양에 의한 RFLP와 비배양에 의한 DGGE 분석 방법에 의해 세균군집 구조를 조사하였다. 16S rDNA-RFLP 분석을 위해 변형된 Zobell 배지와 MA를 이용하여 120균주를 선별하고 제한효소, HaeIII와 MspI을 사용하여 각각의 다른 RFLP 패턴으로 구분하였다. RFLP 패턴으로부터 유래한 16S rDNA 염기서열 분석결과, 알려진 세균 종과 94% 이상의 유사도를 나타내었으며 Alphaproteobacteria, Gammaproteobacteria, Actinobacteria, Bacteroidetes, Firmicutes, 5개의 문이 관찰되었다. 그 중 Gammaproteobacteria가 우점하였다. 같은 해면, A. simplex의 DGGE 분석을 위해 total genomic DNA로부터 16S rDNA를 증폭하여 DGGE fingerprinting을 수행한 결과 12개의 서로 다른 밴드가 관찰되었다. 각 밴드의 16S rDNA 염기서열은 알려진 세균의 염기서열과 90% 이상의 유사성을 나타내었으며 대부분의 염기서열은 uncultured bacteria에 속하였다. DGGE 분석으로부터 미생물의 군집은 Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Deltaproteobacteria, Actinobacteria, Chloroflexi, Nitrospira, 7개의 문으로 나타났다. RFLP와 DGGE 방법에 의해 Alphaproteobacteria, Gammaproteobacteria, Actinobacteria가 공통적으로 발견되었으나 전체적인 공생세균의 군집구조는 분석방법에 따른 차이를 나타내었다. 배양에 의한 방법보다 비배양 방법에서 더 다양한 세균군집구조를 나타내었다.

• 대한수의학회지
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• 제41권2호
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• pp.157-165
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• 2001

Amplified fragment length polymorphism(AFLP) technique is based on the polymorphism detection through selective PCR amplification of restriction fragments from digested genomic DNA and thus includes the procedures of the total DNA digestion by endonucleases, ligation of adapters to the ends of the fragments, and following the selective amplification of the restricted DNA fragments. This study were aimed to : (1) determine the genetic variability of S aureus strains, (2) estimate genetic diversity within and among these strains, (3) compare phylogenetic relationships among these strains as genetic markers using AFLP techniques. Genomic DNA was digested with a particular combination of three restriction enzymes with specific recognition sites and the DNA fragments were ligated to restriction specific adapters and amplified using the selective primer combinations. In the S aureus strain, the number of scorable AFLP bands detected per each primer combination varied from 29 to 102, with an average of 61.59 using 27 primer combinations. A total of 1,663 markers were generated, 904 bands of which were polymorphic, showing a 33.48% level of polymorphism with these primer combinations. Among the primer combinations, E02/T02, E02/T03, E04/H02, E02/T01 and E04/H03 primer combinations showed a high level of polymorphism with 0.78, 0.76, 0.74, 0.71 and 0.70, respectively. But T03/H01, E01/T02 and E01/T03 primer combinations showed a low level of polymorphism with 0.38, 0.37 and 0.15, respectively, Therefore, the former primer combinations will be the most effective for AFLP analysis of S aureus. In SA1 sub-types the level of polymorphism of S aureus KCTC 1927 was similar to that of S aureus CU 01(0.825) and higher than those of other strains such as S aureus CU 02 (0.715), S aureus KCTC 2199(0.625), S aureus KCTC 1916(0.607) and S aureus KCTC 1621 (0.553). In SA2 sub-types the level of polymorphism of S aureus CU 07 was similar to that of S aureus CU 08(0.935) and higher than those of both S aureus CU 04(0.883) and S aureus CU 05(0.883) and lower than those of S aureus CU 03(0.583). In SA3 subtypes the level of polymorphism of S aureus CU 11 was similar to that of S aureus CU 12(0.913) and lower than that of S aureus CU 15(0.623). The results proved that AFLP marker analysis of S aureus strain could be used to study the epidemiology of mastitis and in addition, common genotype in geographic region could be useful for the development of an effective vaccine or DNA marker for easy diagnosis of mastitis caused by S aureus infection.

• Journal of Microbiology and Biotechnology
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• 제14권3호
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• pp.599-606
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• 2004

Gram-positive antagonistic bacilli were isolated from agricultural soils for possible use in biocontrol of plant pathogenic fungi, Fusarium oxysporum, Rhizoctonia solani, and/or Pythium ultimum. Among the 65 antagonistic Gram-positive soil isolates, 22 strains were identified as Bacillus species by 16S rDNA sequence analyses. Four strains, including DF14, especially exhibited multiple antagonistic properties against the three damping-off fungi. Genotypic properties of the Bacillus isolates were characterized by rapid molecular fingerprinting methods using repetitive extragenic palindromic-PCR (REP-PCR), ribosomal intergenic spacer-length polymorphisms (RIS-LP), 16S rDNA PCR-restriction fragment length polymorphisms (PCR-RFLP), and strain-specific PCR assays. The results indicated that the REP-PCR method was more valuable than the RIS-LP and 16S rDNA PCR-RFLP analyses as a rapid and reliable approach for bacilli typing and identification. The use of strain-specific primers designed based on 16S rDNA sequence comparisons enabled it to be possible to selectively detect a strain, DF14, which is being used as a biocontrol agent against damping-off fungi.

• 한국동물위생학회지
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• 제32권3호
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• pp.257-264
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• 2009

Subtyping of Brucella abortus isolates is epidemiologically important for monitoring of bovine brucellosis outbreaks. Pulsed-field gel electrophoresis (PFGE) is considered as a gold standard of molecular typing methods to study the DNA polymorphisms of bacteria. In this study, we analyzed using PFGE the DNA fragment profiles of B. abortus isolated in Gyeongbuk province from 1998 to 2006. The genomic DNA was digested with the restriction endonuclease Xba I, Xho I and Smi I followed gel electrophoresis. No distinguishable patterns of the genomic DNA digested with Xba I and Xho I were observed among the field isolates of B. abortus tested in this study. But Smi I restriction enzyme resulted in two PFGE patterns consisting of 13-15 bands that ranged in size from 33 to 668bp by standard marker. The cluster analysis by DNA fingerprinting software showed 93.75% similarity between two PFGE patterns. No different PFGE patterns were recognized among the isolates originated from various years, regions and cow breeds.

• Asian-Australasian Journal of Animal Sciences
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• 제13권8호
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• pp.1040-1043
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• 2000

The genomic mapping of Red Jungle Fowl (Gallus gallus), local Village Chicken, and broiler was carried out by random amplified polymorphism DNA (RAPD) technique. Two different sets of arbitrary primers were used (Operon OPA01-20 and Genemed GM01-50). All the genomes of the three species of chickens were amplified with OPA01-20 primers. The genomes of the Red Jungle Fowl and local Village Chicken were further amplified with GM01-50 primers. Analysis of the results based on band sharing (BS) and the molecular size of individually amplified DNA fragments showed that Red Jungle Fowl and local Village Chicken shared the species similarity of 66% with Operon primers 01-20, 64% between local Village Chicken and broiler, and 63% when DNA bands between Red Jungle Fowl and broiler were compared. With GM01-50, the BS between Red Jungle Fowl and local village chicken increased to 72%. The results showed that the local village chicken is more closely related to Red Jungle Fowl than to broiler in the genetic distance. On the other hand, broiler is 1% closer in genetic distance to local village chicken than to Red Jungle Fowl. The results also indicated that primers like OPA-7, 8 and 9 can be used as species specific DNA markers for these three species of chickens.