• International Journal of Oral Biology
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• 제46권3호
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• pp.140-145
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• 2021

This study aimed to develop Lautropia mirabilis-specific quantitative real-time polymerase chain reaction (qPCR) primers based on the sequence of DNA-directed RNA polymerase subunit beta gene. The PrimerSelect program was used in designing of the qPCR primers, RTLam-F4 and RTLam-R3. The specificity of the qPCR primers were performed by conventional PCR with 37 strains of 37 oral bacterial species, including L. mirabilis. The sensitivity of the primers was determined by qPCR with the serial dilution of purified genomic DNA of L. mirabilis KCOM 3484, ranged from 4 ng to 4 fg. The data showed that the qPCR primers could detect only L. mirabilis strains and as little as 40 fg of genome DNA of L. mirabilis KCOM 3484. These results indicate that this qPCR primer pair (RTLam-F4/RTLam-R3) may be useful for species-specific detection of L. mirabilis in epidemiological studies of oral bacterial infectious diseases such as periodontal disease.

• 한국축산식품학회지
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• 제27권2호
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• pp.209-215
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• 2007

본 연구는 mt DNA 12S rRNA 유전자의 PCR-RFLP 분석기법을 이용하여 다양한 식육자원 및 각종 가공 육제품의 원료육에 대한 정확하고 재현성 높은 축종 및 육종 감별기술을 개발하기 위하여 수행되었다. 국내에서 유통되고 있는 9종류 축종(소, 돼지, 양, 염소, 말, 사슴, 닭, 오리 및 칠면조)의 육류로부터 12S rRNA유전자의 특정 염기서열을 포함하는 primer를 설계 제작하여 PCR-RFLP 분석을 실시하였다. 각 공시축의 근육조직으로부터 genomic DNA를 추출하고 PCR 증폭 반응을 수행한 후 얻어진 PCR 증폭산물(약 455 bp)을 Tsp5091와 MboI 제한효소로 각각 절단한 결과 Tsp5091 제한효소는 포유류 6종간에서 그리고 MboI 제한효소는 가금류 3종간에서 명확한 차이를 보이는 종 특이적인 PCR-RFLP profile을 검출하였다. 따라서 본 연구에서 개발한 12S rRNA 유전자의 종 특이적 DNA 분자표지는 각종 원료육 및 가공 육제품의 육종 및 축종 판별에 매우 유용한 동물 종 감별 DNA marker로 이용될 수 있을 것이다.

• IMMUNE NETWORK
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• 제11권5호
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• pp.268-280
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• 2011

Background: Dengue virus, which belongs to the Flavivirus genus of the Flaviviridae family, causes fatal dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) with infection risk of 2.5 billion people worldwide. However, approved vaccines are still not available. Here, we explored the immune responses induced by alternating prime-boost vaccination using DNA vaccine, adenovirus, and vaccinia virus expressing E protein of dengue virus type 2 (DenV2). Methods: Following immunization with DNA vaccine (pDE), adenovirus (rAd-E), and/or vaccinia virus (VV-E) expressing E protein, E protein-specific IgG and its isotypes were determined by conventional ELISA. Intracellular CD154 and cytokine staining was used for enumerating CD4+ T cells specific for E protein. E protein-specific CD8+ T cell responses were evaluated by in vivo CTL killing activity and intracellular IFN-${\gamma}$ staining. Results: Among three constructs, VV-E induced the most potent IgG responses, Th1-type cytokine production by stimulated CD4+ T cells, and the CD8+ T cell response. Furthermore, when the three constructs were used for alternating prime-boost vaccination, the results revealed a different pattern of CD4+ and CD8+ T cell responses. i) Priming with VV-E induced higher E-specific IgG level but it was decreased rapidly. ii) Strong CD8+ T cell responses specific for E protein were induced when VV-E was used for the priming step, and such CD8+ T cell responses were significantly boosted with pDE. iii) Priming with rAd-E induced stronger CD4+ T cell responses which subsequently boosted with pDE to a greater extent than VV-E and rAd-E. Conclusion: These results indicate that priming with live viral vector vaccines could induce different patterns of E protein-specific CD4+ and CD8+ T cell responses which were significantly enhanced by booster vaccination with the DNA vaccine. Therefore, our observation will provide valuable information for the establishment of optimal prime-boost vaccination against DenV.

• IMMUNE NETWORK
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• 제5권2호
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• pp.68-77
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• 2005

Background: Costimulation is a critical process in Ag-specific immune responses. Both B7.1 and CD28 molecules have been reported to stimulate T cell responses during antigen presentation. Therefore, we tested whether Ag-specific immune responses as well as protective immunity are influenced by coinjecting with B7.1 and CD28 cDNAs in a mouse HSV-2 challenge model system. Methods: ELISA was used to detect levels of antibodies, cytokines and chemokines while thymidine incorporation assay was used to evaluate T cell proliferation levels. Results: Ag-specific antibody responses were enhanced by CD28 coinjection but not by B7.1 coinjection. Furthermore, CD28 coinjection increased IgG1 production to a significant level, as compared to pgD+pcDNA3, suggesting that CD28 drives Th2 type responses. In contrast, B7.1 coinjection showed the opposite, suggesting a Th1 bias. B7.1 coinjection also enhanced Ag-specific Th cell proliferative responses as well as production of Th1 type cytokines and chemokines significantly higher than pgD+pcDNA3. However, CD28 coinjection decreased Ag-specific Th cell proliferative responses as well as production of Th1 types of cytokines and chemokine significantly lower than pgD+pcDNA3. Only MCP-1 production was enhanced by CD28. B7.1 coimmunized animals exhibited an enhanced survival rate as well as decreased herpetic lesion formation, as compared to pgD+pcDNA3. In contrast, CD28 vaccinated animals exhibited decreased survival from lethal challenge. Conclusion: This study shows that B7.1 enhances protective Th1 type cellular immunity against HSV-2 challenge while CD28 drives a more detrimental Th2 type immunity against HSV-2 challenge, supporting an opposite role of B7.1 and CD28 in Ag-specific immune responses to a Th1 vs Th2 type.

• 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.

• Animal Systematics, Evolution and Diversity
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• 제38권4호
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• pp.274-278
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• 2022

One marine ciliate, Diophrys quadrinucleata Zhang et al., 2020 was newly recorded from South Korea in this study. We provided morphological diagnosis and images of the Korean D. quadrinucleata population. We determined the small subunit ribosomal DNA (SSU rDNA) and cytochrome oxidase subunit I (CO1) sequence data of D. quadrinucleata, and then the sequences were compared with other Diophrys species. Intra-specific variation between the Korean and type (Chinese) populations was identical in the SSU rDNA, while the inter-specific variations between seven Diophrys species were 0.3-3.8% in the SSU rDNA and 12.6-18.2% in the CO1. In this study, we obtained 18S and CO1 data from species with identified morphology. As the importance of securing 18S and CO1 based on morphology increases in current studies, this study will contribute to ciliate studies.

• 한국응용곤충학회지
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• 제34권2호
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• pp.100-105
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• 1995

정확한 in vitro 전사가 일어날 수 있는 진딧물의 세포추출액을 제조하였다. 전사를 직접 조절할 수 있는 단백질 인자를 규명하기 위하여 전사개시점과 그의 상류에 결합하는 DNA 결합단백질을 탐색했다. 전사개시점을 포함하는 단편 A(-194/23)에는 52kDa, 50kDa, 40kDa의 단백질들이 결합했으며 전사개시점 상류의 DNA 단편 B(-393/-263)에는 52kDa, 50kDa, 40kDa의 단백질들이 결합한 반면 DNA 단편 C(-263/-195)는 53kDa단백질만이 결합했다. 그리고 이들 DNA 결합단백질들의 DNA 결합 활성에는 양이온이 요구되었다.

• 미생물과산업
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• 제14권1호
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• pp.22-28
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• 1988

DNA 클로닝과 조작기술의 발전은 어떤 유전자의 특정한 위치에 선택적으로 돌연변이를 도입할 수 있는 site-specific mutagenesis 기술을 창출해 내었다. 이 기술로 DAN 염기의 치환, 결실, 삽입등을 클론된 유전자에 직접 도입할 수가 있게 되어 생체의 유전자 조작이나 유전자의 산물인 단백질의 구조와 기능을 의도적으로 변화시키는 protein engineering에 광범위하게 이용되고 있다. Protein engineering은 주로 단백질의 촉매 및 생리활성의 증가, 효소의 특성및 기질 특이성의 변화, 단백질 구조의 안정화 및 내염성 증가, 분자량의 감소, 효소및 생리활성 단백질의 구조의 안정화및 내열성 증가 등에 활용되고 있으며 산업적 유용성이 큰새로운 단백질의 창조에도 기여할 것으로 기대를 모으고 있다. Site-specific mutagenesis 기술로 현재 가장 널리 이용되는 것이 in vitro상에서 수행하는 oligonucleotide-directed site specific mutagenesis이다. 이 방법은 생화학적으로 합성한 특정한 염기서열을 가진 oligonucleotide들을 일종의 mutagen으로 사용하거나 효소적 DNA 합성을 위한 primer로 사용하여 클론된 DNA의 염기서열을 선택적으로 개조하거나 혹은 다른 조작을 하는 것이다. 여기서는 돌연변이율을 높이는 여러가지 개량된 방법들이 나왔으며 그중의 몇가지를 소개하였다.

• BMB Reports
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• 제38권1호
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• pp.24-27
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• 2005

The role of 3' exonuclease excision in DNA polymerization was evaluated for primer extension using inert allele specific primers with exonuclease-digestible ddNMP at their 3' termini. Efficient primer extension was observed in amplicons where the inert allele specific primers and their corresponding templates were mismatched. However, no primer-extended products were yielded by matched amplicons with inert primers. As a control, polymerase without proofreading activity failed to yield primer extended products from inert primers regardless of whether the primers and templates were matched or mismatched. These data indicated that activation was undertaken for the inert allele specific primers through mismatch proofreading. Complementary to our previously developed SNP-operated on/off switch, in which DNA polymerization only occurs in matched amplicon, this new mutation detection assay mediated by $exo^+$ DNA polymerases has immediate applications in SNP analysis independently or in combination of the two assays.

• International Journal of Oral Biology
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• 제35권1호
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• pp.21-25
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• 2010

This study was undertaken to develop species-specific forward and universal reverse PCR primers for the detection of Streptococcus sobrinus. These primers target the variable regions of the 16S ribosomal RNA coding gene (rDNA) and their specificity was tested against 10 strains of S. sobrinus strains and 20 different species of oral bacteria using serial dilutions of the purified genomic DNA of S. sobrinus ATCC $33478^T$. Our data show that species-specific amplicons were obtained from all the S. sobrinus strains tested but not from other species. Both direct and nested PCR could detect as little as 400 pg and 4 fg of genomic DNA from S. sobrinus ATCC $33478^T$, respectively. This result suggests that these PCR primers are highly specific and sensitive and applicable to the detection of S. sobrinus.