• Title/Summary/Keyword: RAPD-PCR

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Enhanced Discrimination of Listeria spp. Using RAPD Fingerprinting Complemented by Ribotyping-PCR (리스테리아균의 특성분석을 위한 Molecular Typing 방법의 상호보완)

  • 임형근;홍종해;박경진;최원상
    • Journal of Life Science
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    • v.13 no.5
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    • pp.699-704
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    • 2003
  • The results typed by random amplification of polymorphic DNA (RAPD) were compared with those obtained by Enterobacterial repititive intergenic consensus (ERIC) fingerprinting and ribotyping-PCR. The discriminatory power of RAPD typing was the best among the methods tested. RAPD typing with two different primers for 13 Listeria spp. reference strains produced 11 patterns each. In contrast, ERIC fingerprinting produced 9 patterns and ribotyping-PCR produced 7 patterns each. Composite of two separate RAPD (Lis 11 and primer 6) results or RAPD (Lis11)/ ribotyping-PCR differentiated all 13 Listeria spp. reference strains. Therefore, composite of 2 separate RAPD (Lis11 and primer 6) or composite of RAPD (Lis11)/ribotyping-PCR is expected the most promising approach for typing field isolated Listeria spp. strains.

Optimization of RAPD-PCR Conditions for Onions, Allium cepa L. (양파(Allium cepa L)의 RAPD 분석조건 최적화에 관한 연구)

  • 정순재;양보경;김익수;박선희;서전규;남재성;김현경;김도훈
    • Journal of Life Science
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    • v.10 no.2
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    • pp.182-187
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    • 2000
  • The optimized RAPD-PCR conditions, which can be utilized as a basic information for the analysis of the genetic characteristics were investigated with four onion varieties, named Changryungdaego, Yeoeuijuhwang, Yakwangju, and Dabonghwang using Operon primers, OPR01 (TGCGGGTCCT) and OPZ20 (ACTTTGGCGG). We tested several concentrations of DNA, primer, and MgCl2, annealing temperature, number of PCR cycle, and presence/absence of pre-heating time at the begining of PCR reation in the 25${mu}ell$ volume. The best RAPD profiles were obtained using 50ng of DNA, 5mM of primer, 1.5mM of MgCl2, 45$^{\circ}C$ of annealing temperature and an absence of pre-heating time. An establishment of the stable and reproducible RAPD-PCR conditions are expected to be useful for the subsequent RAPD-related investigation, such as genetic characterization of the onion strains, re-establishment of phylogenetic relationships and development of new varieties.

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Optimization of RAPD-FCR Conditions for Morus alba L. (뽕나무(Morus alba L.)의 RAPD 분석조건 최적화에 관한 연구)

  • 정대수;양보경;김나영;정순재;남재성;이영병;이재헌;김경태;김도훈
    • Journal of Life Science
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    • v.14 no.1
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    • pp.110-114
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    • 2004
  • The optimized RAPD-PCR conditions, that can be utilized as a basic information for analysis of the gelletic characteristics were developed for genetic analysis of four mulberry varieties, named Milsung, Chungil, Suil, and Hansung using a primer, OPY15 (5'-AGTCGCCCTT-3') from Operon company. We tested several different factors for best PCR condition including concentrations of DNA, primer, Mgclu annealing temperature, number of PCR cycle, and prosence/absence of pre-heating time at the begining of PCR reaction in the $25 \mul$volume. The best RAPD profiles were obtained using 50 ng of DNA, 1 $\mu$M of primer, $1 \mum$of $MgCl_2\;,45^{\circ}C$ of annealing temperature and an absence of pre-heating time. An establishment of the stable and reproducible RAPD-PCR conditions are expected to be useful for the subsequent RAPD-related investigation, such as genetic characterization of the mulberry varieties, re-establishment of phylogenetic relationships and development of new varieties.

Genetic Relationship among Three Scallop Species, Chlamys farreri farreri, Patinopecten yessoensis and Agropecten irradians, Using RAPD Markers (RAPD표지인자를 이용한 3종의 가리비에 대한 유전적 유연관계)

  • 지희윤;김윤경;박영재
    • The Korean Journal of Malacology
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    • v.17 no.1
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    • pp.1-6
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    • 2001
  • The genetic relationship was examined with PCR-RAPD markers among three scallop species, Chlamys farreri farreri, Patinopecten yessoensis, and Agropecten irradians. Six primers were selected from 60 primers used to compare PCR-RAPD profiles among species. All primers showed distinct RAPD band patterns between the three species. In Chiamys farreri farreri, the morphological characteristics such as shell size and color were considerably different between the two geographical populations. RAPD profile, however, showed that no significant genetic differences were found between the two geographical populations. Polymorphic alleles were observed within a population of each species. Thus, PCR-RAPD markers are useful in identifying scallop species and in understanding scallop population genetic structure.

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RAPD-PCR Analysis in Fusarium species (Fusarium 종에서의 RAPD-PCR분석)

  • 민병례;양연주;최영길
    • Korean Journal of Microbiology
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    • v.35 no.2
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    • pp.107-114
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    • 1999
  • To assess genetic diversity amoug 21 strains from sixleen Frrsn~i~nn species , we used RAPD(rando1n amplified pol.ymorphic DNA) analysis based on PCR(po1ymerase chain reaction). Eleven primers showing Ule polymorphism were chosen from the 40 random pnmers-tcstcd. A total of 263 polymorphic bands were generated by the primers and the size of amplified DNA fragments ranged from 0.1 lo 3.0 kb. Sirnilku-it), coefficients between strains were calcnlatcd, and UPGMA cluster analysis was used to generate a dendrogram showing relationships among them. The results from RAPD-PCR analysis were grouped into four main groups at the si~nilarity level of 0.627.

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Limits of Direct PCR Amplification from Seaweeds Using Arbitrary and ITS Primers (해조류로부터 Arbitrary 및 ITS Primer들을 사용한 직접 PCR 유전자 증폭반응의 한계)

  • 김용국;진형주;박선미;진덕희;홍용기
    • Journal of Life Science
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    • v.9 no.1
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    • pp.15-21
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    • 1999
  • The random amplified ploymorphic DNAs (RAPD) assay is a simple and useful tool in identification of appropriate genetic markers, that requires no knowledge of target DNA sequence. RAPD products were generated directly from seaweed tissues, without prior nucleic acid extraction, of Porphyra yezoensis, Ulva pertusa and Undaria pinnatifida. The nuclear rDNA internal transcribed spacer (ITS) fragment however was not amplified directly from the seaweed tissues. Using DNA extracted by the LiCl method, both the ITS and RAPD's have been amplified by the polymerase chain reaction. RAPD of P yezoensis, thallus (n) and conchocelis (2n) produced lots of different polymorphic bands (36-50$\%$) depending on the arbitrary primers used. Difference was also observed between direct tissues amplification and DNA extracts amplification (53-57$\%$). Thus it is important to use the same ploidy of tissue for DNA extraction and as a RAPD template.

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Random Amplified Polymorphic DNA-PCR Analysis for Identification of Bacillus anthracis (탄저균의 Random Amplified Polymorphic DNA-PCR 분석)

  • 김성주;박경현;김형태;조기승;김기천;최영길;박승환;이남택;채영규
    • Korean Journal of Microbiology
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    • v.37 no.1
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    • pp.56-60
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    • 2001
  • Molecular typing of Bacillus anthracis has been extremely difficult due to the lack of polymorphic DNA markers. Aiming to develop a DNA marker specific for Bacillus anthracis and to be able to discriminate this species from Bacillus genus, we applied the random amplified polymorphic DNA (RAPD)-PCR. We have identified B. anthracis from various Bacillus species. The analysis performed by RAPD clearly demonstrated substantial genetic variations among Bacillus species. This type of analysis is an easy, quick and highly discriminatory technique that may help in diagnosis of anthrax.

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Molecular Typing of Staphylococcus aureus Strains from Domestic Animals and Humans by REP-PCR Analysis (REP-PCR을 이용한 국내 사람과 동물유래 Staphylococcus aureus 분리주의 Molecular Typing)

  • Woo Yong-Ku;Kim Shin
    • Korean Journal of Microbiology
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    • v.41 no.1
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    • pp.60-66
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    • 2005
  • To select the rapid and efficient molecular subtyping method for epidemiologic monitoring of Staphylococcus aureus (S. aureus) strains at clinical laboratory levels, a total 116 of S. aureus and MRSA (methicillin-resistant S. aureus) strains from diverse animal species [Korean cattle, goat, pig, dog, chicken, mouse] and also humans were analyzed. To evaluate the discriminatory ability (DA) of individual PCR methods, random amplified polymorphic of DNA [RAPD; 4M & RA primer], repetitive extragenic palindromic sequences PCR (REP-PCR), and enterobacterial repetitive intergenic consensus sequences PCR (ERIC-PCR) methods were conducted and then compared on their Simpson's index of diversity (SID) values based on the dendrogram patterns, which was produced by software program (BiolD2+ & GelCompar II). In first, RAPD using the 4M primer (SID 0.915) was expressed more higher SID value than that of RA primer (SID 0.874). 4M primer was expressed more powerful DA than RA. Both REP-PCR (SID 0.930) and ERIC-PCR (SID 0.929) methods showed much more higher DA than that of RAPD. According to the present results, both REP-PCR and ERIC-PCR among the tested analysis methods were found as the most reliable and discriminative molecular subtyping method, because they expressed the highest DA for the present S. aureus and MRSA strains.

Analyses of Genetic Relationships of Collectorichum spp. Isolated from Sweet Persimon with RAPD and PCR-RFLP. (단감나무로부터 분리한 탄저병 병원균 Colletotrichum spp.의 RAPD와 PCR-RFLP를 이용한 유연관계 분석)

  • 김희종;엄승희;이윤수
    • Korean Journal of Microbiology
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    • v.38 no.1
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    • pp.19-25
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    • 2002
  • Colletotrichum species are important fungal pathogen that cause great damages on various host plant species worldwide. In Korea, Colletotrichum species cause massive economic losses on apple, peach, grape, and essecially, sweet persimon productions. In the past, Identification of the pathogen and the studies on the genetic relationships among the pathogenic isolates were mainly based on morphology, cultural characteristics, and the difference in pathogenicity. However, in recent years, these traditional methods have been replaced with molecular methods to solve the difficulty of classification on pathogens. Therefore, in this study, RAPD and PCR-RFLP methods were employed for the studies of genetic relationship among the different isolates of Colletotrichum species that cause damages on sweet persimon. As a results of genetic relationship analysis, Colletotrichum species tested were divided into two big groups or five small groups.

Molecular Typing of Leuconostoc citreum Strains Isolated from Korean Fermented Foods Using a Random Amplified Polymorphic DNA Marker

  • Kaur, Jasmine;Lee, Sulhee;Sharma, Anshul;Park, Young-Seo
    • Food Engineering Progress
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    • v.21 no.2
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    • pp.174-179
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    • 2017
  • For preliminary molecular typing, PCR-based fingerprinting using random amplified polymorphic DNA (RAPD) is the method of choice. In this study, 14 bacterial strains were isolated from different Korean food sources, identified using 16S rRNA gene sequencing, and characterized through RAPD-PCR. Two PCR primers (239 and KAY3) generated a total of 130 RAPD bands, 14 distinct PCR profiles, 10 polymorphic bands, one monomorphic band, and four unique bands. Dendrogram-based analysis with primer 239 showed that all 14 strains could be divided into seven clades out of which clade VII had the maximum of seven. In contrast, dendrogram analysis with the primer KAY3 divided the 14 L. citreum strains into four clades out of which clade IV consisted of a maximum of 10 strains out of 14. This research identified and characterized bacterial populations associated with different Korean foods. The proposed RAPD-PCR method, based on sequence amplification, could easily identify and discriminate the lactic acid bacteria species at the strain-specific level and could be used as a highly reliable genomic fingerprinting tool.