• 한국생물물리학회:학술대회논문집
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• 한국생물물리학회 2001년도 학술 발표회 진행표 및 논문초록
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• pp.22-22
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• 2001

At the eukaryotic replication fork, discontinuous synthesis of lagging strand DNA gives rise to Okazaki fragments carrying ribonucleotides derived from the primer RNA at their 5' ends. Efficient removal of these ribonucleotides is vital for maintaining genome integrity. In this report we show that the endonucleases Dna2 and Fen1 act sequentially to facilitate the complete removal of the primer RNA.(omitted)

• 대한핵의학회지
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• 제28권2호
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• pp.220-226
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• 1994

RT-PCR법에 의한 HCV-RNA검출을 임상검사에 적용하는 노력의 일환으로 biotin 및 $^{125}I$을 표지시킨 primer를 이용하여 최근 개발되어 있는 상품화된 kit 로 HCV-RNA 검출을 시도하고 그 결과를 평가하여 보았다. 연구대상은 anti-HCV 양성인 118명의 성인환자로, 방법은 환자 혈청내의 HCV-RNA를 guanidium-thiocynate 용액을 이용하여 추출한 뒤 AMPLICIS II $HCV^{(R)}$(CIS, France)kit를 이용하여 cDNA로 역전사 후 1차 증폭을 시행하고, 다시 소량의 1차 증폭산물을 biotin과 $^{125}I$으로 각각 표지된 1쌍의 Primer로 이용하여 2차 증폭을 시행하였다. 증폭산물은 avidin이 코팅된 시험관에 반응시키고 감마선 계수기로 계수하였다. 검사의 재현성을 보기 위하여 anti-HCV 양성인 환자 혈청 중 무작위로 51개의 검체를 선정하여 반복실험하였고, 만성 간질환 환자로부터 얻은 34개의 검체는 본 연구소에서 확립된 다른 RT-PCR 방법으로도 검사를 병행하여 비교하였다. 결과는 다음과 같다. 1) Anti-HCV 양성이면서 조직검사상 만성감염이나 간경화 소견을 보이거나 6개월이상 지속적인 간기능 이상을 나타낸 환자 88명 중 85명 (97%)에서 HCV-RNA 양성이었고, 6개월 이상 간기능이 정상이었던 30명중에서는 73%인 22명에서 HCV-RNA양성 이었다. 2) 두가지 다른 방법으로 RT-PCR을 병행한 34명중, kit를 사용한 경우는 32명(94%)에서 양성소견을 보였고, 기존방법대로 전기 영동 후 ethidium bromide로 염색한 경우는 27명 (79%)에서 양성을 나타내었다. 3) 검사의 재현성을 보기 위하여 anti-HCV 양성인 51개의 검체를 2번 반복 실험하였을때, 82%에서 일치된 결과를 얻을 수 있었다. 이상에서 biotin 및 $^{125}I$ 표지된 primer로 구성된 kit를 이용했을 때 기존방법에 비하여 예민하게 HCV-RNA를 검출할 수 있었다. 그러나 여전히 검사에 많은 노력과 시간이 소요되고 반복 검사시 검사간 변이가 상당히 존재하고 있어 정도관리에 세심한 주의가 필요할 것으로 사료되었다.

• BMB Reports
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• 제30권2호
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• pp.162-165
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• 1997

RNA I. a negative controller of ColE1-type plasmid replication, is metabolized by several RNases in Escherichia coli. Two small derivatives of RNA I are accumulated at nonpermissive temperatures in an E. coli strain carrying the rnpA49 mutation, a thermosensitive mutation in the rnpA gene encoding the protein component of RNase P. A primer extension analysis was carried out to compare 5' processing of RNA I in the E. coli rnpA49 cells at both permissive and nonpermissive temperatures. Derivatives of RNA I having different 5' ends were observed in the cells grown at permissive and nonpermissive temperatures. Some of the derivatives may be generated by the cleavage of RNase P.

• Parasites, Hosts and Diseases
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• 제37권4호
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• pp.257-263
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• 1999

Identification of the genes responsible for the recovery of virulence in brain-passaged Acanthamoeba culbertsoni was attempted via mRNA differential display polymerase chain reaction (mRNA DD-PCR) analysis. In order to identify the regulatory changes in transcription of the virulence related genes by the brain passages, mRNA DD-PCR was performed which enabled the display of differentially transcribed mRNAs after the brain passages. Through mRNA DD-PCR analysis. 96 brain-passaged amoeba specific amplicons were observed and were screened to identify the amplicons that failed to amplify in the non-brain-passaged amoeba mRNAs. Out of the 96 brain-passaged amoeba specific amplicons, 12 turned out to be amplified only from the brain-passaged amoeba mRNAs by DNA slot blot hybridization. The clone, A289C, amplified with an arbitrary primer of UBC ＃289 and the oligo dT$_{11}$-C primer, revealed the highest homology (49.8％) to the amino acid sequences of UPD-galactose lipid transferase of Erwinia amylovora, which is known to act as an important virulence factor. The deduced amino acid sequences of an insert DNA in clone A289C were also revealed to be similar to cpsD, which is the essential gene for the expression of type III capsule in group B streptococcus. Upregulated expression of clone A289C was verified by RNA slot blot hybridization. Similar hydrophobicity values were also observed between A289C (at residues 47-66) and the AmsG gene of E. amylovora (at residues 286-305: transmembrane domains). This result suggested that the insert of clone A289C might play the same function as galactosyl transferase controlled by the AmsG gene in E. amylovora.a.

• 식물병연구
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• 제14권3호
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• pp.223-225
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• 2008

2005년 전북 고참에서 채집한 옥수수 이병주로부터 벼검은줄오갈병 바이러스를 동정하였다. 이들 이병주로부터 게놈 dsRNA를 추출하여 polyacrylamide gel 전기영동으로 게놈 패턴을 분석 하였다. 전기영동 결과 이미 알려진 10개의 분절게놈을 확인하였으며 채집지역별 isolate에서 게놈 dsRNA이동도의 차이를 확인하였다. 추출된 dsRNA를 주형으로 하여 S10의 full-length 특이 primer를 사용하여 RT-PCR한 결과 1,801의 예상되는 band를 확인하여 RBSDV로 동정하였다. S10을 pGEM-T vector에 크로닝하여 염기서열 분석 결과 1,801nt, 559aa로 구성되어 있었다. 이는 벼에 발생하는 RBSDV S10의 크기와 동일하였으며 상동성 분석결과 18개 염기에서 변이가 확인되어 99%의 상동성을 나타내었다.

• Journal of Microbiology and Biotechnology
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• 제17권3호
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• pp.490-495
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• 2007

Lactic acid bacteria (LAB) are beneficial for the gastrointestinal tract and reinforce immunity in human health. Recently, many functional products using the lactic acid bacteria have been developed. Among these LAB, Lactobacillus acidophilus, Lactobacillus rhamnosus, Bifidobacterium longum, and Bifidobacterium bifidum are frequently used for probiotic products. In order to monitor these LAB in commercial probiotic products, a multiplex PCR method was developed. We designed four species-specific primer pairs for multiplex PCR from the 16S rRNA, 16S-23S rRNA intergenic spacer region, and 23S rRNA genes in Lactobacillus acidophilus, Lactobacillus rhamnosus, Bifidobacterium longum, and Bifidobacterium bifidum. Using these primer pairs, 4 different LAB were detected with high specificity in functional foods. We suggest that the multiplex PCR method developed in this study would be an efficient tool for simple, rapid, and reliable identification of LAB used as probiotic strains.

• The Plant Pathology Journal
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• 제24권3호
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• pp.279-282
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• 2008

This study describes a phytoplasmal disease occurring in Petunia leaves grown in the glasshouse of the National Horticultural Research Institute, Suwon, Korea. Abnormal growth like flattened stem with flower malformation or phyllody was observed from the plant. The DNA extracted from the diseased leaves was amplified using a universal primer pair of P1/P6 derived from the conserved 16S rRNA gene of Mollicutes giving the expected polymerase chain reaction(PCR) product of 1.5 kb. In the nested PCR assays, the expected DNA fragment of 1.1 kb was amplified with the specific primer pair R16F1/R16R1 that was designed on the basis of aster yellows(AY) phytoplasma 16S rDNA sequences. The 1.1 kb PCR products were cloned and nucleotide sequences were determined, and the sequences of the cloned 168 rRNA gene were deposited in the GenBank database under the accession no. of EU267779. Analysis of the homology percent of the 168 rDNA of PFS-K showed the closest relationship with Hydrangea phyllody phytoplasma(AY265215), Brassica napus phytoplasma(EU123466) and AY phytoplasma CHRY(AY180956). Phytoplasma isolated from the diseased Petunia was designated as Petunia flat stem phytoplasma Korean isolate(PFS-K) in this study. Flattened stem occurring in Petunia was confirmed as infection of AY group of phytoplasma by determination of 16S rRNA gene sequences of phytoplasma and microscopic observation of phytoplasma bodies. This is the first report on the phytoplasmal disease in Petunia in Korea.

• 한국어병학회지
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• 제10권1호
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• pp.39-44
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• 1997

Metallothionein은 세균에서 척추동물에 이르기 까지 모든 생명체에 존재하며, 중금속의 세포내농도를 조절하는 중요한 단백질이다. 현재까지 metallothionein의 기능 및 유도기작에 관한 연구는 많이 진척되지는 않았으나, 여러 metallothionein 유전자의 구조가 밝혀져 있는 실정이다. 특히 어류의 metallothionein은 여러종류의 중금속과 환경적인 자극에 의하여 유도되고 정량적인 RT-PCR의 방법으로 metallothionein 유전자의 RNA transcript를 측정함으로써 환경적인 자극의 정도와 중금 속의 상대적인 양을 측정할 수 있기 때문에 중요한 단백질로 인식되고 있다. 본 연구에서는 유전자내부의 특이적 primer와 통상적인 3`말단의 primer를 이용하여 PCR에 의해 450 bp에 해당하는 metallothionein 유전자의 일부의 특성을 조사하였다. 챠넬메기의 cDNA library로부터 PCR에 의해 증폭된 450 bp의 PCR 절편은 다른 어류의 metallothionein 유전자와는 유사성을 보이지 않았다.

• Journal of Microbiology and Biotechnology
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• 제20권2호
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• pp.245-253
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• 2010

The base sequences representing human- and cow-specific 168 rRNA gene markers identified in a T-RFLP analysis were recovered from clone libraries. The human- and cow-specific primers were designed from these sequences and their specificities were analyzed with fecal DNAs from human, cow, and pig. The AllBac primer set showed positive results for all human, cow, and pig samples, whereas the human-specific primer set showed positive result only for the human sample but not for the cow or pig samples. Likewise, the cow-specific primer set showed positive results only for the cow sample but not for the human or pig samples. Real-time PCR assay with these primers was developed for the identification and quantification of fecal pollution in the river water. The human- and cow-specific markers were detected in the order of 9 $\log_{10}$ copies per gram wet feces, which were two orders of magnitude lower than those of total Bacteroidales. For the river water samples, the human-specific marker was detected in $1.7-6.2\;\log_{10}$ copies/100 ml water, which was 2.4-4.9 orders of magnitude lower than those of total Bacteroidales. There was no significant correlation between total Bacteroidales and conventional fecal indicators, but there was a high correlation between Bacteroidales and the human-specific marker. This assay could reliably identify and quantify the fecal pollution sources, enabling effective measures in the watersheds and facilitating water quality management.

• 한국축산식품학회지
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• 제33권6호
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• pp.696-700
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• 2013

A quick and reliable method for identifying meat origin is developed to ensure species origin of livestock products for consumers. The present study examined the identification of meat sources (duck, chicken, goat, deer, pig, cattle, sheep, and horse) using PCR by exploiting the mitochondrial 12S rRNA and mitochondrial cytochrome b genes. Species-specific primers were designed for some or all mitochondrial 12S rRNA nucleotide sequences to identify meat samples from duck, chicken, goat, and deer. Mitochondrial cytochrome b genes from pig, cattle, sheep, and horse were used to construct species-specific primers, which were used to amplify DNA from different meat samples. Primer sets developed in this study were found to be superior for detecting meat origin when compared to other available methods, for which the discrimination of meat origin was not equally applicable in some cases. Our new development of species-specific primer sets could be multiplexed in a single PCR reaction to significantly reduce the time and labor required for determining meat samples of unknown origin from the 8 species. Therefore, the technique developed in this study can be used efficiently to trace the meat origin in a commercial venture and help consumers to preserve their rights knowing origin of meat products for social, religious or health consciousness.