• Biotechnology and Bioprocess Engineering:BBE
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• 제11권3호
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• pp.235-239
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• 2006

Due to recent advances in genome sequencing, there has been a dramatic increase in the quantity of genetic information, which has lead to an even greater demand for a faster, more parallel expression system. Therefore, interest in cell-free protein synthesis, as an alternative method for high-throughput gene expression, has been revived. In contrast to in vivo gene expression methods, cell-free protein synthesis provides a completely open system for direct access to the reaction conditions. We have developed an efficient cell-free protein synthesis system by optimizing the energy source and S30 extract. Under the optimized conditions, approximately $650{\mu}g/mL$ of protein was produced after 2h of incubation, with the developed system further modified for the efficient expression of PCR-amplified DNA. When the concentrations of DNA, magnesium, and amino acids were optimized for the production of PCR-based cell-free protein synthesis, the protein yield was comparable to that from the plasmid template.

• 식물조직배양학회지
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• 제21권6호
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• pp.319-326
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• 1994

십자화과 식물의 유용유전자를 cloning하기 위한 기초연구로서 십자화과 식물인 Armoracia rusicna의 재분화계와 형질전환계를 확립하고, gene tagging을 하기 위하여 binary vector에 삽입된 옥수수의 transposon 유전자 Ac/Ds를 도입한 결과, NAA 0.1 mg/L와 BA 1.0 mg/L를 함유한 MS 배지에서 최적의 shoot를 유기할 수 있었으며, MS 기본배지에 옮기면 쉽게 발근을 유도할 수 있었다. 옥수수의 Ac/Ds의 유전자를 잎에 형질전환시킨 결과 8-10%의 형질전환율을 보였으며, 엽병의 경우에도 4%의 형질전환 식물체가 얻어졌다. Kanamycin 100 mg/L 농도에서 선발한 개체를 PCR 분석 및 Southern blot분석을 행하였던 결과 PCR분석으로부터 Ds유전자가 식물에 도입된 것이 확인되었고, Southern blot 분석으로부터 Ac/Ds 모두가 도입된 것이 확인되었다.

• Journal of Genetic Medicine
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• 제1권1호
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• pp.39-43
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• 1997

The mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) is inherited maternally, in which the MTTL1*MELAS 3243 mutation has been most commonly found as a heteroplasmy of A to G point mutation in the $tRNA^{Leu(UUR)}$ gene. The MTTL1*MELAS 3271 mutation is known to be the second common mutation, though clinical features of both mutations are not remarkably different. Recently, a variety of minor mutations have been reported in patients with MELAS. In this study, major efforts have been made to investigate the allele frequency of major three mutations including MTTL1*MELAS 3243, 3252, 3271 in 10 Korean families with MELAS probands. The PCR and subsequent direct sequencing of the PCR product in the regions spanning these three mutation sites were employed to identify the mutation in each proband. All family members have been screened for the presence of these three mutations by PCR-RFLP assay using Apa I, Acc I and Bfr I restriction enzymes. The MTTL1*MELAS 3243 mutation was most commonly found (7 out of 10 families tested) followed by the MTTL1*MELAS 3271 which was identified in 1 out of 10 families. In the remaining 2 families none of three mutations were found, indicating the presence of either nuclear mutation or yet unidentified mitochondrial DNA mutation in these families.

• The Plant Pathology Journal
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• 제15권3호
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• pp.137-143
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• 1999

A novel chromosomal gene tagging technique using a specific fragment of the fatty acid desaturase-like open reading frame (des-like ORF) from the tox-argK gene cluster of Pseudomonas syringae pv. phaseolicola was developed to identify Xanthomonas spp.released into the environment as biocontrol agents. X. campestris pv. convolvuli FB-635, a pathogen of Convolvulus arvensis L., (bindweed), was chosen as the organism in which to develop and test the system. A 0.52 kb DES fragment amplified from P. syringae pv. phaseolicola C-199 was inserted into pGX15, a cosmid clone containing a 10.3 kb Eco RI-HindIII fragment derived from the xanthomonadin biosynthetic gene cluster contained in plasmid pIG102, to create a pigG::DES insertion. The 10.8 kb EcoRI-BamHI fragment carrying the pigG:: DES insertion was cloned into pLAFR3 to generate pLXP22. pLXP22 was then conjugated into X. campestris pv. convolvuli FB-635 and the pigG::DES insertion integrated into the bacterial chromosome by marker exchange. Rifampicin resistant, tetracycline sensitive, starch hydrolyzing, white colonies were used to differentiate the marked strain from yellow pigmented wild-type ones. PCR primers specific for the unique DES fragment were used for direct detection of the marked strain. Result showed the marked strain could be detected at very low levels even in the presence of high levels of other closely related or competitive bacteria. This PCR-based DES-tagging system provides a rapid and specific tool for directly monitoring the dispersal and persistence of Xanthomonas spp.released into the environment.

• The Plant Pathology Journal
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• 제34권4호
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• pp.286-296
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• 2018

Maintenance of a beneficial microbial community, especially in the rhizosphere, is indispensable for plant growth and agricultural sustainability. In this sense, plant growth-promoting rhizobacteria (PGPR) have been extensively studied for their role in plant growth promotion and disease resistance. However, the impact of introducing PGPR strains into rhizosphere microbial communities is still underexplored. We previously found that the Proteus vulgaris JBLS202 strain (JBLS202) promoted growth of Kimchi cabbage and altered the relative abundance of total bacteria and Pseudomonas spp. in the treated rhizosphere. To extend these findings, we used pyrosequencing to analyze the changes in bacterial communities in the rhizosphere of Kimchi cabbage after introduction of JBLS202. The alterations were also evaluated by taxon-specific realtime PCR (qPCR). The pyrosequencing data revealed an increase in total bacteria abundance, including specific groups such as Proteobacteria, Acidobacteria, and Actinobacteria, in the treated rhizosphere. Time-course qPCR analysis confirmed the increase in the abundance of Acidobacteria, Actinobacteria, Alphaproteobacteria, and Betaproteobacteria. Furthermore, genes involved in nitrogen cycling were upregulated by JBLS202 treatment indicating changes in ecological function of the rhizosphere soil. Overall, these results indicate that introduction of JBLS202 alters both the composition and function of the rhizosphere bacterial community, which can have direct and indirect effects on plant growth. Therefore, we propose that long-term changes in bacterial composition and community-level function need to be considered for practical use of PGPRs.

• 대한수의학회지
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• 제56권3호
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• pp.147-153
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• 2016

A total of 782 blood and 465 tissue samples from 1,039 wild animals and 127 dairy goats were collected from January 2011 to December 2013 in 10 provinces of South Korea and tested for the presence of brucellosis. The Rose Bengal test revealed that 8.0% (52/650) of the serum samples were seropositive, while 4.2% (33/782) of the serum samples were positive for Brucella antibodies by competitive enzyme-linked immunosorbent assay. Of the 650 sera examined, only 16 (2.5%) were positive by both serological tests. Direct polymerase chain reaction (PCR) assay using B4/B5 primers for Brucella abortus (BCSP31) revealed the prevalence of Brucella to be 26.5% (129/487) in blood samples and 21% (98/465) in tissue samples while, 16S rRNA PCR detected Brucella DNA in 6.8% (33/487) and 2.6% (12/465) in blood and tissue samples, respectively. Of PCR-positive samples, only 6.2% (30/487) of blood samples and 2.4% (11/465) of tissue samples were found to be positive by both BCSP31 and 16S rRNA PCRs. However, Brucella strains were isolated by blood culture from only two out of 487 blood samples (0.4%). This characterization and identification of pathogenic Brucella isolates is the first to clearly indicate that the organisms were Brucella abortus biovar 1.

• Restorative Dentistry and Endodontics
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• 제28권2호
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• pp.178-183
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• 2003

The purpose of this study was to investigate the frequency of 7 putative pathogens in endodontic infections. The specimens were collected from infected pulpal tissue of patients who were referred for root canal treatment to the department of conservative dentistry, Chosun University Samples were collected aseptically using a barbed broach and a paper point. The cut barbed broaches and paper points were transferred to an eppendorf tube containing 500 ml of 1 X PBS. DNAs were extracted from the samples by direct DNA extraction method using lysis buffer (0.5% EDTA, 1% Triton X-100). Identification of 7 putative pathogens was performed by PCR based on 16S rDNA. The target species were as follows : Porphyromonas endodontalis, Porphyromonas gingivalis, Prevotella intermedia, Prevotella nigrescens, Bacteroides forsythus, Actinobacillus actinomycetemcomitans, and Treponema denticola. Our data revealed that the prevalence of P. endodontalis was found in 88.6% (39/54), P. ginivalis 52.3% (23/44), P. nigrescens 18.2% (8/44), P intermedia 15.9% (7/44) B. forsythus 18.2% (8/44), A. actinomycetemcomitans 3.3% (1/44), T. denticola 25% (l1/44) of the samples. The high prevalence of P. endodontalis and P. ginivalis suggests that they may play an important role in the etiology of endodontic infections.

• Tuberculosis and Respiratory Diseases
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• 제50권2호
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• pp.222-228
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• 2001

목 적 : 객담 항산균 도말 양성환자에서 direct amplification test (DAT) 양성이면 폐결핵으로 확진이 가능하다. 그러나 객담 항산균 도말음성이고 DAT는 양성인 경우에는 DAT 결과가 얼마나 신뢰성이 있는지 의문이었다. 따라서 저자들은 폐결핵이 의심되는 환자에서 객담 항산균 도말 음성이고 DAT 양성일 때 폐결핵진단에 있어서 DAT의 유용성올 살펴보고자 하였다. 방 법 : 1998년 6월1일부터 1999년 5월30일 까지 한림대학교 의료원에서 폐결핵이 의심되어 결핵균 항산균 도말, 배양검사 및 DAT를 시행한 909명을 대상으로 하였다. 활동성 폐결핵의 확진은 결핵균 배양 양성이거나 조직학적으로 건락성 육아종이 증명된 경우로 정의하였다. 임상적 폐결핵의 진단 조건은 방사선 소견상 결핵성 폐침윤이 의심되고 폐렴, 폐암 등이 배제된 환자로 결핵균 배양은 음성이나, 항결핵제 3개월 이상 투여 후 증상 및 방사선 소견상 호전이 있는 경우로 정의하였다. DAT는 Mycobacterium tuberculosis complex에 특징적으로 존재 하는 insertion sequence (IS6110)의 특정 부위만을 특이적으로 증폭시킬 수 있는 TB-$CRkit^{TM}$(한국생공, 대전)를 이용하였으며, 특이도를 높이기 위해 nested PCR검사를 시행하였다. 결 과 : 대상 환자 909 명 중 객담 항산균 도말검사는 335명에서 양성(36.6%)이었으며, 객담 결핵균 PCR이 374명에서 양성(41.1%)으로 판명되었다. 객담 항산균 도말 음성이면서 객담 결핵 PCR 양성인 환자는 39명(4.3%)으로 남자 22명, 여자 17명 이었다. 객담 결핵 PCR 양성인 환자 39 명중 32명에서 폐결핵으로 진단되었다. DAT는 확진된 결핵환자만을 대상으로 할 때 61.5%의 양성 예측율을, 확진된 폐결핵 환자와 임상적으로 진단된 결핵환자를 대상으로 할 때 82.1% 양성 예측율을 나타냈었고 위양성율은 18.0%였다. 결 론 : 이상의 결과로 임상적으로 폐결핵이 의심되는 객담 항산균 도말 음성인 환자에서 객담 DAT 양성일 때 폐결핵의 조기 진단에 중요한 수단으로 이용 될 수 있을 것으로 사료되었다.

• Pediatric Infection and Vaccine
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• 제22권2호
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• pp.106-112
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• 2015

목적: 장기간의 항생제 치료는 중이염 어린이 환자의 중이액으로부터 원인균이 배양되는 것을 방해한다. 본 연구는 배양 음성 중이액으로부터 분자적 진단에 의한 신속한 균 검출 가능성 여부를 확인하고자 하였다. 방법: 폐구균 lytA 유전자를 표적으로 하는 PCR과 LAMP로 민감도와 특이도를 비교 결정하고, 임상중이액에서의 폐구균 검출에 적용하였다. 결과: PCR 기법에 의한 폐구균 검출 최소한계는 약 $10^4$ 집락형성단위(CFU)이고, LAMP의 검출 최소한계는 10 CFU에서 결정되었다. 한편 두 가지 검사법 모두 Haemophilus influenzae 와 Moraxella catarrhalis 에 대해 $10^6$ CFU 이상에서도 DNA를 증폭하지 않았다. 22개의 배양음성 중이액 중에서 12개 검체가 LAMP-양성(54.5%, 12/22)으로 확인되었고, 이들 12개 LAMP-양성 검체 중, 3개의 검체만이 PCR-양성으로 확인되었다(25%, 3/12). 본 연구의 결과는 LAMP 기법의 폐구균 검출 해상력이 PCR 기법에 비교하여 4배 이상 높음을 보여준다(P<0.01). 결론: lytA -특이 LAMP 기법은, 중이액 내의 타 병원균과는 교차반응 없이 10 CFU 폐구균의 DNA를 검출할 수 있는 고해상 기술로서, 중이액 폐구균 검출 및 폐구균 백신의 보급에 따른 백신 효과 평가에 적용이 기대된다.

• 대한바이러스학회지
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• 제29권4호
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• pp.271-281
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• 1999

The nested polymerase chain reaction (PCR) assay was used to determine the sequences of reverse transcriptase (RT) codons 41, 67, 70, 210, 215 and 219 of human immunodeficiency virus-1 (HIV-1) pol gene. Template DNA was obtained from uncultured peripheral blood mononuclear cells from 27 Korean HIV-1 infected patients treated with ZDV and Korean red ginseng. The second PCRs were done for 2 separated regions (RT codons $13{\sim}98$ and $152{\sim}259$) with $5\;{\mu}l$ of the first PCR productNucleotide sequences were determined by direct sequencing. In the 27 patients, CD4+ cell count decreased from $230{\pm}117/{\mu}l$ to $152{\pm}162/{\mu}l$ for $46{\pm}26$ months (Mo), and actual duration of ZDV intake was $72{\pm}16$ Mo. In the 16 patients who had been treated with ZDV therapy ${\ge}25$ Mo, the incidences of 70R, 215F/Y, and 41L were 61%, 28% and 22%, respectively and those of 67N, 210W and 219Q were 17%. The incidences of 215F/Y were 6.7% for group ${\le}12$ Mo treatment, 22.7% for group with 13 to 24 Mo, and 27.8% for group ${\ge}25$ Mo. There was no mutation in 9 patients. It might be associated with the interruption of ZDV therapy for more than 6 months in 6 patients. This study shows that the detection of mutation could be useful prognostic marker with other clinical and virological data, and very low mutation rate is dectected compared to overseas reports.