• Title/Summary/Keyword: DNA separation

검색결과 121건 처리시간 0.021초

한국 재래품종과 외래품종의 구별을 위한 초위성체 마커의 개발 (Development of Microsatellite Markers for Discriminating Native Korean and Imported Cattle Breeds)

  • 김승창;조창연;노희종;연성흠;최성복
    • 생명과학회지
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    • 제27권4호
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    • pp.464-470
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    • 2017
  • 성염색체에 위치하는 5 개의 초위성체 마커(INRA30, TGLA325, UMN0803, UMN0905, UMN0929) 를 이용하여 재래소 3품종과 외래소 7품종(칡소, 한우, 제주흑우, 홀스타인, 일본화우, 샤롤레, 앵거스, 헤어포드, 시멘탈, 한우X 샤롤레 교잡종)의 유전적 특징을 확인하였다. 상업적으로 판매되는 소고기의 잘못된 원산지 표기를 통해 부당한 경제적 이득을 취하고자 하는 문제를 해결하기 위한 방법으로 소고기 샘플을 빠르고 저비용으로 확인 하기 위한 방법으로 사용하기 위해 좌위 또는 품종 특이적 대립유전자를 탐색하고 좌위별 대립유전자수, 대립유전자빈도, 이형접합도 그리고 다형정보량(PIC)을 구하여 이들 10품종의 유전적 다양성을 평가하였다. STRUCTURE 분석을 통한 군락의 분류 및 유전적 균일성 분석에서 재래소 품종과 외래소 품종으로 두개의 주요 그룹으로 나뉘어진다. 이러한 결과들은 재래소와 외래소 품종의 특이적인 유전적 차이를 나타낸다. 또한 Nei's 표준 유전적 거리로 나타난 neighbor-joining tree에서도 독립적인 계통유전학적인 위치를 보여주었다. 이러한 결과는 국내 재래종과 외래품종 사이의 유전적 거리, 품종의 역사 및 그들의 지리적 기원 사이에 명백한 차이를 나타내는 증거로 사료된다. 이러한 결과들로 이들 성염색체의 초위성체 마커들에 의해 소 품종들의 유전적 다양성과 연관성은 과학적인 기초자료로 활용되고 재래소와 외래품종 소고기를 구별할 수 있는 DNA 마커들로 이용될 수 있을 것으로 사료된다. 그러므로 이러한 마커들은 효율적인 이력추적 시스템을 만드는데 사용되어 원산지 표시 위반을 억제하는데 유용할 것이다.

Flammulina velutipes var. lupinicola의 유전체 정보기반 laccase 유전자 동정 및 특성 규명 (Identification and characterization of laccase genes in the Flammulina velutipes var. lupinicola genome)

  • 유혜원;박영진
    • 한국버섯학회지
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    • 제19권4호
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    • pp.285-293
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    • 2021
  • 본 연구에서는 Flammulina velutipes var. lupinicola의 laccase 유전자를 동정하고 최적 활성 pH, 온도, 시간을 분석하고 하였다. F. velutipes var. lupinicola 유전체에서 선별된 laccase 유전자 서열을 바탕으로 구리 결합 부위 및 신호 펩타이드 분석을 수행한 결과 5종의 laccase 유전자(g1934, g1937, g2415, g2539, g5858)를 동정하였다. 5종의 선별된 laccase 유전자 크기는 1,488~1,662 bp로 확인되었고, cDNA 염기서열 분석 결과 14~17개의 인트론이 확인되었다. Laccase 유전자의 신호펩타이드로 예측된 절단 부위는 N-말단으로부터 20~34 bp 사이에 위치하는 것으로 확인되었다. F. velutipes var. lupinicola laccase의 활성 특성을 규명하기 위해 분리 정제를 수행하였으며, Zymogram을 수행하여 0.2 M 및 0.3 M NaCl과 1.6 M 및 1.7 M의 ammonium sulfate로 정제된 단백질에서 5개의 laccsae 활성 밴드를 확인하였다. pH, 온도 및 시간별로 분리 정제된 단백질의 최적 활성을 분석한 결과, 반응의 최적 pH는 5.5이고 최적 온도는 40℃로 확인되었다. 따라서 본 연구를 통하여 확인된 F. velutipes var. lupinicola 유전체의 laccase 유전자 구조 및 활성에 대한 특성은 laccase를 이해하는 데 도움이 될 것이며 추가 연구를 통하여 향후 다양한 산업적 활용이 가능할 것으로 사료된다.

Detection of Campylobacter jejuni in food and poultry visors using immunomagnetic separation and microtitre hybridization

  • Simard, Ronald-E.
    • 한국어업기술학회:학술대회논문집
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    • 한국어업기술학회 2000년도 춘계수산관련학회 공동학술대회발표요지집
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    • pp.71-73
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    • 2000
  • Campylobacter jejuni is most frequently identified cause of cause of acute diarrhoeal infections in developeed countries, exceeding rates of illness caused by both salmonella and shigilla(Skirrow, 1990 ; Lior 1994). Previous studies on campylobacter jejuni contamination of commercial broiler carcasses in u.s.(Stern, 1992). Most cases of the disease result from indirect transmission of Campylobactor from animals via milk, water and meat. In addition to Campylobactor jejuni. the closely relates species Campylobactor coli and Campylobactor lari have also been implicated as agents of gastroenteritis in humans. Campylobactor coli represented only approximately 3% of the Campylobactor isolates from patients with Campylobactor enteritis(Griffiths and Park, 1990) whereas Campylobactor coli is mainly isolated from pork(Lmmerding et al., 1988). Campylobactor jejuni has also been isolated from cases of bacteremia, appendicitis and, recently, has been associated with Guillai-Barre syndrome(Allos and Blaser, 1994; von Wulffen et al., 1994; Phillips, 1995). Studies in volunteers indicated that the infectious dose for Campylobactor jejuni is low(about 500 organisms)(Robinson, 1981). The methods traditionally used to detect Campylobactor ssp. in food require at least two days of incubation in an enrichment broth followed by plating and two days of incubation on complex culture media containing many antibiotics(Goossens and Butzler, 1992). Finnaly, several biochemical tests must be done to confirm the indentification at the species level. Therfore, sensitive and specific methods for the detection of small numbers of Campylobactor cells in food are needed. Polymerase chain reaction(PCR) assays targeting specific DNA sequences have been developed for the detection of Campylobactor(Giesendorf and Quint, 1995; Hemandex et al., 1995; Winter and Slavidk, 1995). In most cases, a short enrichment step is needed to enhance the sensitivity of the assay prior to detection by PCR as the number of bacteria in the food products is low in comparison with those found in dinical samples, and because the complex composition of food matrices can hinder the PCR and lower its sensitivity. However, these PCR systems are technically demanding to carry out and cumbersome when processing a large number of samples simutaneously. In this paper, an immunomagnetic method to concentrate Campylobactor cells present in food or clinical samples after an enrichment step is described. To detect specifically the thermophilic Campylobactor. a monoclonal antibody was adsorbed on the surface of the magnetic beads which react against a major porin of 45kDa present on the surface of the cells(Huyer et al., 1986). After this partial purification and concentration step, detection of bound cells was achieved using a simple, inexpensive microtitre plate-based hybridization system. We examined two alternative detection systems, one specific for thermophilic Campylobactor based on the detection of 23S rRNA using an immobilized DNA probe. The second system is less specific but more sensitive because of the high copy number of the rRNA present in bacterial cell($10^3-10^4$). By using specific immunomagnetic beads against thermophilic Campylobactor, it was possible to concentrate these cells from a heterogeneous media and obtain highly specific hybridization reactions with good sensitivity. There are several advantages in using microtitre plates instead of filter membranes or other matrices for hybridization techniques. Microtitre plates are much easier to handle than filter membranes during the adsorption, washing, hybridization and detection steps, and their use faciilitates the simultanuous analysis of multiple sample. Here we report on the use of a very simple detection procedure based on a monoclonal anti-RNA-DNA hybrid antibody(Fliss et al., 1999) for detection of the RNA-DNA hybrids formed in the wells.

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Evaluation of the Diversity of Cyclodextrin-Producing Paenibacillus graminis Strains Isolated from Roots and Rhizospheres of Different Plants by Molecular Methods

  • Vollu Renata Estebanez;Fogel Rafael;Santos Silvia Cristina Cunha dos;Mota Fabio Faria da;Seldin Lucy
    • Journal of Microbiology
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    • 제44권6호
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    • pp.591-599
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    • 2006
  • To address the diversity of cyclodextrin-producing P. graminis strains isolated from wheat roots and rhizospheres of maize and sorghum sown in Australia, Brazil, and France, restriction fragment length polymorphism analysis of part of genes encoding RNA polymerase (rpoB-RFLP) and DNA gyrase subunit B (gyrB-RFLP) was used to produce genetic fingerprints. A phylogenetic tree based on rpoB gene sequences was also constructed. The isolates originated from Brazil could be separated from those from Australia and France, when data from the rpoB-based phylogenetic tree or gyrB-RFLP were considered. These analyses also allowed the separation of all P. graminis strains studied here into four clusters; one group formed by the strains GJK201 and $RSA19^T$, second group formed by the strains MC22.02 and MC04.21, third group formed by the strains TOD61, TOD 221, TOD302, and TOD111, and forth group formed by all strains isolated from plants sown in Cerrado soil, Brazil. As this last group was formed by strains isolated from sorghum and maize sown in the same soil (Cerrado) in Brazil, our results suggest that the diversity of these P. graminis strains is more affected by the soil type than the plant from where they have been isolated.

Modified T-RFLP Methods for Taxonomic Interpretation of T-RF

  • Lee, Hyun-Kyung;Kim, Hye-Ryoung;Mengoni, Alessio;Lee, Dong-Hun
    • Journal of Microbiology and Biotechnology
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    • 제18권4호
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    • pp.624-630
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    • 2008
  • Terminal restriction fragment length polymorphism (T-RFLP) is a method that has been frequently used to survey the microbial diversity of environmental samples and to monitor changes in microbial communities. T-RFLP is a highly sensitive and reproducible procedure that combines a PCR with a labeled primer, restriction digestion of the amplified DNA, and separation of the terminal restriction fragment (T-RF). The reliable identification of T-RF requires the information of nucleotide sequences as well as the size of T-RF. However, it is difficult to obtain the information of nucleotide sequences because the T-RFs are fragmented and lack a priming site of 3'-end for efficient cloning and sequence analysis. Here, we improved on the T-RFLP method in order to analyze the nucleotide sequences of the distinct T-RFs. The first method is to selectively amplify the portion of T-RF ligated with specific oligonucleotide adapters. In the second method, the termini of T-RFs were tailed with deoxynucleotides using terminal deoxynucleotidyl transferase (TdT) and amplified by a second round of PCR. The major T-RFs generated from reference strains and from T-RFLP profiles of activated sludge samples were efficiently isolated and identified by using two modified T-RFLP methods. These methods are less time consuming and labor-intensive when compared with other methods. The T-RFLP method using TdT has the advantages of being a simple process and having no limit of restriction enzymes. Our results suggest that these methods could be useful tools for the taxonomic interpretation of T-RFs.

Optimization of SNP Genotyping Assay with Fluorescence Polarization Detection

  • Cai Chun Mei;Van Kyujung;Kim Moon Young;Lee Suk-Ha
    • 한국작물학회지
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    • 제50권5호
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    • pp.361-367
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    • 2005
  • Single nucleotide polymorphisms (SNPs) are valuable DNA markers due to their abundance and potential for use in automated high-throughput genotyping. Numerous SNP genotyping assays have been developed. In this report, one of effective and high throughput SNP genotyping assays, which was named the template-directed dye-terminator incorporation with fluorescence polarization detection (FP-TDI) was described. Although the most of this assay succeed, the objective of this work was to deter­mine the reasons for the failures, find ways to improve the assay and reduce the running cost. Ninety $F_2$-derived soybean, Glycine max (L.) Merr., RILs from a cross between 'Pureunkong' and 'Jinpumkong 2' were genotyped at four SNPs. FP measurement was done on $Victot^3$ microplate reader (perkinelmer Inc., Boston, MA, USA). Increasing the number of thermal cycles in the single-base extension step increased the separation of the FP values between the products corresponding to different genotypes. But in some assays, excess of heterozygous genotypes was observed with increase of PCR cycles. We discovered that the excess heterozygous was due to misincorporation of one of the dye­terminators during the primer extension reaction. After pyrophosphatase incubation and thermal cycle control, misincoporation can be effectively prevented. Using long amplicons instead of short amplicons for SNP genotyping and decreasing the amount of dye terminator and Acyclopol Taq polymerase to 1/2 or 1/3 decreased the cost of the assay. With these minor adjustments, the FP-TDI assay can be used more accurately and cost-effectively.

Otolith microchemistry reveals the migration patterns of the flathead grey mullet Mugil cephalus (Pisces: Mugilidae) in Korean waters

  • Bae, Seung Eun;Kim, Jin-Koo
    • Journal of Ecology and Environment
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    • 제44권3호
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    • pp.185-195
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    • 2020
  • Background: The flathead grey mullet Mugil cephalus has the widest distribution among mugilid species. Recent studies based on mitochondrial DNA sequences showed that the species comprises at least 14 different groups, three of which occur in the northwest Pacific. We analyzed the otolith microchemistry of M. cephalus at several locations in Korea to improve understanding of migration pattern and population origin. Results: We collected 123 sagittal otoliths from seven locations and determined their concentrations of eight elements (7Li, 24Mg, 55Mn, 57Fe, 60Ni, 63Cu, 88Sr, and 138Ba) using laser ablation inductively coupled plasma mass spectrometry. Mean otolith elemental ratios differed significantly among the locations. The Sr:Ca, Fe:Ca, and Ba:Ca ratios were significantly higher than others, and useful chemical signatures for investigating the habitat use of M. cephalus populations. We identified five diverse and complicated migration patterns using the otolith data that we collected: estuarine resident (type I), freshwater migrant (type II), estuarine migrant (type III), seawater resident (type IV), and seawater migrant (type V). A canonical discriminant analysis plot revealed separation of two groups (type II in the Yellow Sea vs. other types in remaining locations). Two locations on Jeju Island, despite their close proximity, had fish with quite different migration patterns, corroborating previous molecular studies that distinguished two groups of fishes. Conclusion: We successfully showed that the migration patterns of the Korean mullet varied by location. Only fish from the western sector of Jeju had a unique migration pattern, which is likely confined population in this area. Among the eight otolith elements measured, the Sr:Ca ratio was found to be the best indicator of migration pattern and population origin.

Comparison of Soil Bacterial Community Structure in Rice Paddy Fields under Different Management Practices using Terminal Restriction Fragment Length Polymorphism (T-RFLP)

  • Kim, Do-Young;Kim, Chang-Gi;Sohn, Sang-Mok;Park, Sang-Kyu
    • Journal of Ecology and Environment
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    • 제31권4호
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    • pp.309-316
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    • 2008
  • To develop a monitoring method for soil microbial communities in rice paddy fields, we used terminal restriction fragment length polymorphism (T-RFLP) to compare soil bacterial community structure in rice paddy fields experiencing different management practices: organic practices, conventional practices without a winter barley rotation, and conventional practices with a winter barley rotation. Restriction fragment length profiles from soils farmed using organic practices showed very different patterns from those from conventional practices with and without barley rotation. In principal component analyses, restriction fragment profiles in organic practice samples were clearly separated from those in conventional practice samples, while principal component analysis did not show a clear separation for soils farmed using conventional practices with and without barley rotation. The cluster analysis showed that the bacterial species compositions of soils under organic practices were significantly different from those under conventional practices at the 95% level, but soils under conventional practice with and without barley rotation did not significantly differ. Although the loadings from principal component analyses and the Ribosomal DNA Project II databases suggested candidate species important for soils under organic farming practices, it was very difficult to get detailed bacterial species information from terminal restriction fragment length polymorphism. Rank-abundance diagrams and diversity indices showed that restriction fragment peaks under organic farming showed high Pielou's Evenness Index and the reciprocal of Simpson Index suggesting high bacterial diversity in organically farmed soils.

Screening of Domain-specific Target Proteins of Polo-like Kinase 1: Construction and Application of Centrosome/Kinetochore-specific Targeting Peptide

  • Ji, Jae-Hoon;Jang, Young-Joo
    • BMB Reports
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    • 제39권6호
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    • pp.709-716
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    • 2006
  • Mammalian polo-like kinase 1 (Plk1) acts at various stages in early and late mitosis. Plk1 localizes at the centrosome and maintains this position through mitosis. Thereafter Plk1 moves to the kinetochore and midbody region, important sites during chromosome separation and cytokinesis. The catalytic domain of Plk1 is in the N-terminus region, whereas the non-catalytic region in the C-terminus of Plk1 has a conserved motif, named the Polobox. This motif is critical for Plk localization. EGFP proteins fused with the N-terminus and C-terminus of Plk1 localize in the nucleus and centrosomes, respectively. The core sequences of the polo-box (50 amino acids) also localize in Plk1 target organelles. To screen for domain-specific target proteins of Plk1, we constructed an N-terminal domain and a tandem repeat polo-box motif, and used them as templates in a yeast two-hybrid screen. The HeLa cell cDNA library indicated several proteins including the centrosome/kinetochore components or regulators, to be characterized as positive clones. Through in vitro protein binding analyses, we confirmed an interaction between these proteins and Plk1. The data reported from this study indicate that the N- and C- termini of Plk1 may function through recruitment and/or activation of domain-specific target proteins in dividing cells. Additionally, tandem repeats of the conserved core motif of the polo-box are sufficient for targeting and may be useful as a centrosome/kinetochore-specific targeting peptide.

RAPD 방법을 이용한 거제도, 개도, 제도해역에서 채집한 말잘피 개체분석 (Population analysis of eelgrass, Zostera marina L. in Geojedo, Gaedo, and Jedo on the southern coastal water of Korea using RAPD-PCR)

  • 조은섭;이상용;김정배
    • 생명과학회지
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    • 제17권4호
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    • pp.455-461
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    • 2007
  • 본 연구는 말잘피 군집의 유전적 구조 및 다양성 분석은 잘피 관리를 위해서 시행했다. 거제도, 개도, 제도연안에 서식하는 잘피를 대상으로 RAPD분석에 따른 유전적 다양성이 높은 것으로 나타났다. 거제도는 개도 및 제도와는 100 km 이상 떨어진 지역에 서식하는 잘피의 유전적 거리는 0.16으로 나타난 반면에,개도와 제도의 유전적 거리는 0.08정도로 보이고 있다. 거제도에 서식하는 잘피의 개체내 유전적 유사도는 70% 이상으로 나타났으나, 개도와 제도에 비하면 낮은 유사도를 보이고 있다. 따라서 남해안에 서식하고 있는 잘피의 개체는 지역적 거리에 따라 유전적으로 상이한 집단을 형성하고 있다. 이러한 원인은 시스트 확산 및 유전자 이동률 제한 때문인 것으로 추정된다.