• KSBB Journal
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• 제10권3호
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• pp.264-270
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• 1995

Trigonopsis variabilis는 버l타락탐 항생제인 cephalosporin C (ceph C)를 ${\alpha}$-keto-adipyl-7 a aminocephalosporanic acid(AKA-7 ACA)로 생변 환하는 강력한 D-amino acid oxidase 효소를 갖고 있다. 본 연구는 이 D-AAO 효소의 유전자를 추출하기 위하여 polymerase chain reaction (PCR)을 사용하였다. PCR 단편을 콜로닝하기 위하여 Taq D DNA polymerase, Klenow, T4 DNA polymerase I, Alkaline phosphatase Calf Intestinal와 T4 kinase 의 효소반응을 이용하여 4가지의 방법을 샤용한 결 과, blunt - end 의 PCR fragment 를 phosphory­l lation하고 blunt -end의 pUC18 plasmid를 dephos phorylation 한 후 ligation 한 것 이 가장 좋은 클로 닝 효율을 보였다. Ceph C에 대한 D-AAO 효소의 활성은 재조합 E. coli의 세포추출액과 permea bilized cells에서 모두 확인할 수 있었다.

• Journal of Microbiology and Biotechnology
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• 제9권4호
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• pp.381-385
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• 1999

Taq DNA polymerase from Thermus aquaticus has been shown to be very useful in a polymerase chain reaction. Taq DNA polymerase has a domain at the amino terminus (residues 1 to 290) that has 5'-3' exonuclease activity and a domain at the C-terminus that catalyzes the polymerase reaction. Taq DNA polymerase is classified into the Pol I family, which is represented by E. coli DNA polymerase I. The alignment of amino acid sequences for the 5'-3' exonuclease domains of the Pol I family DNA polymerases shows ten highly conserved carboxylic amino acids. Crystallographic studies suggested that six of the carboxylic amino acids are clustered within a 7 $\AA$ radius by chelating three metal ions in the active site. Those six carboxylic residues are mutagenized to alanines in order to better understand their function. All six carboxylic residues, Asp l8, Glu1l7, Asp1l9, Asp120, Asp142, and Aspl44, are crucial for catalysis of 5'-3' exonuclease.

• 한국임상수의학회지
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• 제14권2호
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• pp.177-184
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• 1997

Canine parvovirus(CPV) is a very highly contagious virus causing hemorrhagic enteritis and myocarditis mainly in young dogs. The diseases were first recognized in 1978, and then spread throughout the world by 1980. The main source of the infection seems to be the feces of infected dogs, at the same time feces are suitable materials for detection of virus in the enteric form exactly for the same reasons. Recently, a new technique of in vitro DNA amplification, Known as the polymerase chain reaction (PCR), has been widely applied to clinical viral diagnosis because of its sensitivity, specificity and rapidity. In this research, we attemped to set up the PCR for the detection of CPV in fecal samples and conformed the canine parvpviral enteritis by PCR. To increase the sensitivity and specificity of a PCR, the nested PCR (two-step PCR) was performed. We also surveyed the contamination status of CPV in the research using fecal specimen was highly sensitive and specific. Of the 100 fecal specimens suspected canine parvoviral enteritis, 45 fecal specimens were positive in HA test, 64 fecal specimens were positive in the first PCR, and 87 fecal specimens were positive in the second PCR. CPV contamination status of animal clinics and breeding centers was serious, wo hygienic management of environment in which dogs are reared is required. The nested PCR described here seems to be a rapid, sensitive and specific for the detection of canine parvovirus.

• 대한임상검사과학회지
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• 제47권2호
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• pp.83-89
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• 2015

결핵(Mycobacterium tuberculosis, MTB) 감염은 아직까지 전 세계에서 높은 유병률과 사망의 주요 원인이 되고 있으며 비정형 결핵(nontuberculous mycobacteria, NTM)은 최근 후천성 면역결핍증(AIDS)이나, 종양, 이식 등으로 면역력이 저하된 환자들의 임상 검체에서 분리빈도가 증가함에 따라 분리 균주의 임상적 의의가 중요시 되고 있다. 이에 항산균 염색을 이용한 객담 도말검사는 결핵균과 비정형 결핵균을 구별할 수 없다는 제한점이 있고, 균 분리배양검사는 시간이 오래 걸린다는 단점이 있어, 본 연구에서는 이러한 제한점을 가진 기존의 검사법을 대신하여 분자생물학적 방법인 중합효소 연쇄반응(polymerase chain reaction, PCR)을 이용하여 균 분리배양 검사와 비교하여 보았다. Mycobacteria를 동정하는데 항산균 염색과 3% ogawa 배지를 이용하였고, 균 분리배양 후 M. tuberculosis를 확인하기 위해서 niacin test를 실시한 결과 집락의 DNA를 추출하여 PCR후 동정된 M. tuberculosis와 niacin 양성이 일치함을 확인할 수 있었다. 또, 이 실험에서 항산성균 염색이 2+ 이상인 객담검체와 집락검체 각각에서 DNA를 추출하여 결핵균을 동정하는 방법으로 M. tuberculosis complex에 특징적으로 존재하는 insertion sequence (IS) 6110의 특정부위인 547 bp와 285 bp 부분을 증폭한 two-tube nested polymerase chain reaction을 시행하였고, 비정형 결핵균 동정법으로는 mycobacteria에 공통적으로 존재하는 rpoB 유전자 중 일부인 360 bp 부분을 증폭한 후, 제한효소 Msp I을 첨가 PCR-restriction fragment length polymorphism (RFLP)을 시행하였으며, 결핵균과 비정형 결핵균 모두 동정율에 거의 차이가 없었다. 1+인 경우는 객담검체에서 PCR한 결과가 31.2%에서 집락검체의 PCR한 결과 93.7%까지 결핵균과 비정형결핵균의 동정율이 높아졌고, trace인 경우 객담검체에서 PCR 결과가 2%에서 집락검체에서 PCR한 결과가 97.9%까지 결핵균과 비정형 결핵균의 동정율을 높일 수 있었다. 이 실험에서 항산성균 염색 1+이하 일때 객담검체와 집락검체로 PCR을 실시하면 결핵균과 비정형 결핵균의 동정율에 차이가 있고, 배양만으로는 결핵균의 동정은 가능하지만 비정형 결핵균의 동정이 가능하지 않으므로 배양검사와 PCR 검사 모두를 병행하므로써 보다 신속하고 정확한 검사결과를 내는데 도움 될 것이다.

• Journal of Microbiology
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• 제37권3호
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• pp.154-158
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• 1999

The arginine residue at position 243 (Arg 243) of the yeast transcription factor, Gcn4p, is invariably conserved among bZIP transcription factors. Using site-directed oligonucleotide saturation mutagenesis involving two-step polymerase chain reaction (PCR) amplification, random mutations were successfully introduced at the codon of 243 in the basic domain of Gcn4p. This mutant library was transformed ito Gcn4p defective yeast strain and selected for the transcriptionally active colonies. All colonies which were transcriptionally active had arginines in the codon 243. In this study, the strand preference by Taq polymerase during mutagenesis was also tested. Oligonucleotides were specially designed to test whether or not the polymerase was preferred using the strand as a template. A population of randomly mutated products were cloned into an appropriate vector and characterized by DNA sequencing analysis. Saturation mutagenesis which was performed efficiently by this method revealed a strong bias in terms of strand preference of Taq polymerase by an approximate ratio of 3 to 1 in this study.

• 한국동물위생학회지
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• 제34권1호
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• pp.13-18
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• 2011

The purpose of this study was to apply a nested-polymerase chain reaction (nPCR) assay to detect and differentiate PCV 2a and PCV 2b. The compared with nPCR and one-step PCR and nPCR showed more sensitive in the detection of PCV-2 from tissue and blood samples. The total of 52 tissue samples was collected from postweanning pigs from 2006 to 2010. All tissue samples showed positive for PCV-2 in one-step PCR and nPCR, followed by the nPCR in order to identify the genotypes of PCV-2. 2 samples (3.8%) showed positive for PCV 2a, and 35 samples were positive for PCV 2b (67.3%), 15 samples (28.9%) were positive the dual genotypes. In addition, 42 blood samples which were collected from the 5 different swine farms were compared figure out the detection rates of nPCR and one-step PCR. The PCV 2 was positive by one-step PCR in 21 samples (50.0%) and nPCR was positive in 37 samples (88.1%). The PCV 2 genotypes in blood samples and 32 samples (76.2%) were positive for PCV 2b and none were positive for PCV 2a, 5 samples (11.9%) were positive for dual genotypes. These results suggest that the nPCR is very efficient for genotyping blood samples and differentiating the genotypes of PCV-2 from field samples.

• 대한수의학회지
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• 제38권1호
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• pp.101-106
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• 1998

The present study attempted to apply polymerase chain reaction (PCR) to develop a rapid differential diagnosis among Marek's disease, reticuloendotheliosis and avian leukosis. The primers chosen to detect Marek's disease virus (MDV) flank the 132bp tandem direct repeat of the MDV genome. The primers selected for reticuloendotheliosis virus (REV) and avian leukosis virus (ALV) are based on proviral long terminal repeats of spleen necrosis virus and Rous-associated virus-2 genomes, respectively. The specific PCR products of MDV, REV and ALV were observed with each primer and the reaction was not cross-reacted among the viruses. MDV-specific DNA was also amplified from the MDV-induced lymphoma (MDCC-MSB1) but not from the REV-induced tumor and ALV-induced lymphoma (LSCC-1104B1). In addition, proviral DNA of REV from REV-induced tumor and proviral DNA of ALV from ALV-induced lymphoma were also amplified by REV-specific and ALV-specific PCRs, respectively. Therefore these three PCR methods may be used to rapidly differentiate among MDV, REV and ALV-associated tumors in diagnosis.

• 한국식품영양과학회지
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• 제27권6호
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• pp.1166-1172
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• 1998

A method for the isolation and purification of DNA from a red algae, Porphyra was innovated. The innovation of the method consists mainly of three steps that include sodium acetate treatment, chloroform extraction, and 0.2 volume isopropanol precipitation step. The sodium acetate treatment was designed to remove polysaccharide contamination, and the isopropanol step to remove proteins and salts contaminents. Genomic DNA,s of several species(for example, P. tenera, P. yezoensis, P. seriata, and P. pseudolinearis) was successfully isolated by the innovated method. The amount of DNA purified from one g of sample material with the innovated method was 53 g in average. The resulting DNA was characterized to include high molecular weight and showed no nuclease activity. The DNA was pure enough to be digested directly by various restriction enzymes without any difficulties. Porphyra DNA was pure enough and adequate for amplification reaction through the polymerase chain reaction (small nuclear rDNA PCR amplification).

• 한국식품영양학회지
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• 제13권1호
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• pp.1-5
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• 2000

Detection for heat-labile enterotoxin(LT) of enterotoxigenic Escherichia coli(ETEC, EC81, O148:H28) and enterotoxin of enterotoxigentic Clostridium perfringents type A(CP, NCTC8238, Hobbs serotype 2) by use of a polymerase chain reaction (PCR) assay were positive reaction, which using LT gene-specific primers of ETEC with a detection limit equivalent from 100ng/${\mu}\ell$ to 1 pg of a DNA fragment of 417-bp in EC81 and enterotoxin gene-specific primers of CP with a detection limit equivalent from 100ng/${\mu}\ell$ to 10pg of a DNA fragment of 364-bp in NCTC8238. Detection for a LT gene of ETEC highly appeared 10-fold sensitivity than an enterotoxin gene of CP.

• Clinical and Experimental Reproductive Medicine
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• 제21권3호
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• pp.281-284
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• 1994

Sex determination in genomic DNA from human blood leucocytes was performed by amplification of human Y chromosome-specific DNA sequences using PCR technique. A clear DNA fragment(154 nucleotides long) was appeared only in the male genomic DNA, but no specific band was observed in the case of female genomic DNA and negative control. To know the sensitivity of this method, the amplification reaction was performed in genomic DNA diluted to 2pg equivalent to the amonut present in the single human cell, and clear band also observed. The PCR amplification was so succesfully performed in the single leucocyte separated from human blood using micromanipulator that this techniqe is assumed to be applied to single blstomere before embryo transfer.