• Asian-Australasian Journal of Animal Sciences
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• 제20권12호
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• pp.1887-1894
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• 2007

In this paper, a rapid and reliable gene-targeted species-specific polymerase chain reaction (PCR) technique based on a two-step process was established to identify bifidobacteria in dairy products. The first step was the PCR assay for genus Bifidobacterium with genus specific primers followed by the second step, which identified the species level with species-specific primer mixtures. Ten specific primer pairs, designed from nucleotide sequences of the 16-23S rRNA region, were developed for the Bifidobacterium species including B. angulatum, B. animalis, B. bifidum, B. breve, B. catenulatum, B. infantis, B. longum, B. minimum, B. subtile, and B. thermophilum. This technique was applied to the identification of Bifidobacterium species isolated from 6 probiotic products, and four different Bifidobacterium spp. (B. bifidum, B. longum, B. infantis, and B. breve) were identified. The findings indicated that the 16S-23S rDNA gene-targeted species-specific PCR technique is a simple and reliable method for identification of bifidobacteria in probiotic products. PCR combined with Denaturing Gradient Gel Electrophoresis (DGGE) for identification of the bifidobacteria was also evaluated and compared with the gene-targeted species-specific technique. Results indicated that for fermented milk products consistency was found for both species-specific PCR and PCR-DGGE in detecting species. However, in some lyophilized products, the bands corresponding to these species were not visualized in the DGGE profile but the specific PCR gave a positive result.

• Asian-Australasian Journal of Animal Sciences
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• 제19권6호
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• pp.775-778
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• 2006

Sarabi cows (n = 136) from the Sarabi Breeding Station were genotyped at bovine lymphocyte antigen (BoLA)-DRB3.2 locus by a genotyping system that used the polymerase chain reaction and restriction fragment length polymorphism. Genomic DNA was extracted from whole blood samples. A two-step polymerase chain reaction was carried out in order to amplify a 284 base-pair fragment of target gene. Nested-PCR products were digested with three restriction endonuclease enzymes RsaI, BstYI and HaeIII. Digested fragments were analyzed by polyacrylamide gel electrophoresis. Twenty-six BoLA-DRB3.2 alleles were identified with frequencies ranging from 0.4 to 15.1%. Six new allele types observed in this study have not been reported previously. Identified alleles include: BoLA-DRB3.$2^*1$, $^*2$, $^*4$, $^*6$, $^*8$, $^*12$, $^*13$, $^*14$, $^*15$, $^*16$, $^*17$, $^*23$, $^*24$, $^*25$, $^*28$, $^*32$, $^*34$, $^*35$, $^*36$, $^*37$, $^*42$, $^*46$, $^*51$, $^*kba$, $^*laa$ and $^*vaa$. Their frequencies were found to be 0.4, 0.4, 0.7, 11.4, 1.1, 1.8, 2.9, 2.2, 4.4, 9.6, 1.1, 13.6, 0.4, 0.4, 1.1, 0.7, 0.4, 6.2, 2.2, 3.7, 1.1, 7.7, 1.5, 15.1, 2.6 and 7.3% respectively. The six most frequent alleles (DRB3.2 $^*6$, $^*16$, $^*23$, $^*46$, $^*kba$ and $^*vaa$) accounted for 64.7% of the alleles in the population of this herd. Numerous studies on this locus, covering different breeds, has revealed the existence of various alleles in this locus, and new investigations have introduced novel alleles. With respect to the high number of the observed alleles in this survey and the novelty of some alleles with no previous record of reporting, it is plausible to conclude that the BoLA-DRB3.2 locus is highly polymorphic in Iranian native Sarabi cows.

• BMB Reports
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• 제38권6호
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• pp.690-694
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• 2005

Transcription termination of the human mitochondrial genome requires specific binding to termination factor mTERF. In this study, mTERF was produced in E. coli and purified by two-step chromatography. mTERF-binding DNA sequences were isolated from a pool of randomized sequences by the repeated selection of bound sequences by gel-mobility shift assay and polymerase chain reaction. Sequencing and comparison of the 23 isolated clones revealed a 16-bp consensus sequence of 5'-GTG$\b{TGGC}$AGANCCNGG-3' in the light-strand (underlined residues were absolutely conserved), which nicely matched the genomic 13-bp terminator sequence 5'-$\b{TGGC}$AGAGCCCGG-3'. Moreover, mTERF binding assays of heteroduplex and single-stranded DNAs showed mTERF recognized the light strand in preference to the heavy strand. The preferential binding of mTERF with the light-strand may explain its distinct orientation-dependent termination activity.

• Journal of Microbiology
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• 제34권3호
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• pp.229-235
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• 1996

Microgram quantities of DNA per gram soil were recovered with SDS- based and freeze-and thaw procedures. The average DNA fragment size was > 23 Kb. This method generated minimal shearing of extracted DNA. However, the DNA extracts still contained considerable amounts of humic impurities sufficient to inhibit PCR. Several approaches were used to reduce the interferences with the PCR (use of CTAF in extraction step, Elutip-d column purification, addition of BSA to PCR buffer) to accomplish PCR with DNA extract as a template. Most of the DNA extracts were not digested completely by restriction endonuclease, and CTAB-TREATED ane Elutip-d column purified DNA extracts were partially digested. Regarding as restriction enzyme digestion, all PCRs failed to amplify 16S rRNA gene fragments in the DNA extracts. In the case of DNA extracts only where BSA was added to PCR buffer, PCR was successfully conducted whether the DNA extracts were treated with CTAB or purified with columns. However, these two treatments were indispensable for humic impurity-rich DNA extracts to generate the PCR-compatible DNA samples. Direct extraction of DNA, coupled with these procedures to remove and relieve interferences by humic impurities and followed by the PCR, can be rapid and simple method for molecular microbiological study on soil microorganisms.

• 한국어병학회지
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• 제31권2호
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• pp.71-79
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• 2018

2015년 2월부터 2018년 8월까지 총 1,253 마리의 자연산 명태 (Gadus chalcogrammus)를 강원도 고성 아야진항 근해에서 정치망을 사용하여 포획한 후, 바이러스 (viral hemorrhagic septicemia virus, VHSV; nervous necrosis virus, NNV; marine birnavirus, MABV) 모니터링을 RT-PCR법으로 수행하였다. One-step PCR법으로 비장시료 및 뇌시료에서는 대상 바이러스가 모두 검출되지 않았으며, 일부 시료를 two-step PCR법으로 검사한 결과 VHSV는 19.7% (36/183)의 비장시료에서 검출되었다. 또한, NNV는 4.4% (8/183)의 비장시료, 1.2% (3/259)의 뇌시료에서 검출되었다. 검출된 바이러스의 계통분석 결과, 기존의 국내에서 분리되는 바이러스의 유전형에 각각 속하는 것으로 나타났다 (Genotype IVa, RGNNV genotype). 바이러스의 분리를 시도하지 않아 검출된 바이러스의 활성은 알 수 없지만, 모든 양성 시료가 two-step PCR법으로 검출되었으므로 매우 낮을 것으로 추측된다.

• 한국수산과학회지
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• 제47권4호
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• pp.370-375
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• 2014

BACE2 is a membrane-bound aspartic protease that is highly homologous with BACE1. While BACE1 processes the amyloid precursor protein (APP) at a key step in generating ${\beta}$-amyloid peptide and presumably causes Alzheimer's disease (AD), BACE2 has not been demonstrated to be involved in APP processing directly, and its physiological functions are unknown. To determine its function and to develop inhibitors from marine sources, we constructed an overexpression vector for producing BACE2. The gene encoding human BACE2 protease was amplified using the polymerase chain reaction and cloned into the pET11a expression vector, resulting in pET11a/BACE2. Recombinant BACE2 protease was overexpressed successfully in E. coli as inclusion bodies, refolded using the rapid-dilution method, and purified via two-step fast protein liquid chromatography using Sephacryl S-300 gel filtration and Resource-Q column chromatography. The BACE2 protease produced was an active form. This study provides an efficient method not only for studying the basic properties of BACE2, but also for developing inhibitors from natural marine sources.

• 한국작물학회지
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• 제52권4호
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• pp.458-468
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• 2007

To tolerate environmentally adverse conditions such as cold, drought, and salinity, plants often synthesize and accumulate proline in cells as compatible osmolytes. ${\Delta}^1$-Pyrroline-5-carboxylate synthetase(P5CS) catalyzes the rate-limiting step of proline biosynthesis from glutamate. Two complete genes, MtP5CS1 and MtP5CS2, were isolated from the model legume Medicago truncatula by cDNA cloning and bacterial artificial chromosome library screening. Nucleotide sequence analysis showed that both genes consisted of 20 exons and 19 introns. Alignment of the predicted amino acid sequences revealed high similarities with P5CS proteins from other plant species. The two MtP5CS genes were expressed in response to high salt and low temperature treatments. Semi-quantitative reverse transcription-polymerase chain reaction showed that MtP5CS1 was expressed earlier than MtP5CS2, indicating differential regulation of the two genes. To evaluate the reverse genetic effects of nucleotide changes on MtP5CS function, a Targeting Induced Local Lesions in Genomes approach was taken. Three mutants each were isolated for MtP5CS1 and MtP5CS2, of which a P5CS2 nonsense mutant carrying a codon change from arginine to stop was expected to bring translation to premature termination. These provide a valuable genetic resource with which to determine the function of the P5CS genes in environmental stress responses of legume crops.

• 생명과학회지
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• 제8권2호
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• pp.181-188
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• 1998

두 종류의 ADP-riboyltransferase(Yac-1과Yac-2)가 최근 mouse의 임파구로부터 cloning되어 그 특성이 규명되어졌다. Yac-1효소는 ADP-ribosyltransferase활성을 보여주나, 대조적으로, Yac-2는 상당한 양의 NAD glycohydrolase 활성도 소유하고` 있으며 이는 NDA를 우선적으로 가수분해 할 수 있다는 사실을 반영한다. Yac-2는 보존된 두 glutamate에 인접한 위치인 219번에 또 다른 glutamate를 소유하고 있다. 효소 활성에 대한 Glu-219의 효과를 알아보기 위해 두 단계의 재조합 중합효소 연쇄 반응 방법에 의해 Glu-219가 glutamate (E219Q)혹은 alanine(E219A)으로 치환되었다. Gln혹은 Ala으로의 치환결과, ADP-ribosyltransferase활성은 56% (E219Q)혹은 66% *E219A)로 감소하였다. Yac-2단백질의 NAD glycohydrolase 활성은 돌연변이에 의해 영향을 받지 않았다. 이러한 결과는 Yac-2효소의 Glu-219가 ADP-ribosyltransferase 활성에 중요한 역할을 하나, NDA glycohydrolase활성에는 관여하지 않음을 시사한다.

• 식물병연구
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• 제26권3호
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• pp.170-178
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• 2020

바이러스의 정확한 진단법 확립은 바이러스의 피해 및 확산을 예방하는데 매우 중요하게 작용한다. 대부분의 딸기 바이러스는 조직내에 낮은 역가로 분포하여 진단이 어렵고, 특히 딸기 조직은 다당류 및 페놀화합물의 함유가 많아 RNA 추출이 어려운 것으로 알려져 있다. 딸기 우량묘 생산에 필요한 바이러스 검정기술을 확립하기 위해 본 연구에서는 딸기 잎에서 바이러스 진단을 위해 가장 최적의 RNA 추출방법 정립을 위해 다양한 상용 키트와 시약을 이용하여 RNA 추출효율 비교하였다. 바이러스 진단을 통한 RNA 추출효율을 분석하기 위해 SMoV 감염주인 미홍 딸기 품종을 이용하여 다양한 단계에서 잎조직으로부터 RNA를 추출하고 바이러스 진단을 수행하였다. 식물 RNA 추출 방법 가운데 상업용으로 판매되는 RNeasy plant mini kit (Qiagen)를 이용하는 경우 본 연구에서 살펴본 one-step 또는 two-step RT-PCR 방법과 무관하게 SMoV의 검출이 잘 되었다. 또한, 딸기 우량묘의 바이러스 검정에 대한 신뢰있는 진단방법을 구축하기 위해 주요 딸기 바이러스인 strawberry mild yellow edge virus (SMYEV), strawberry mottle virus (SMoV), strawberry latent ringspot virus (SLRSV), strawberry crinkle virus (SCV), strawberry pallidosis associated virus (SPaV), strawberry vein banding virus (SVBV) 및 strawberry necrotic shock virus (SNSV) 7종에 대한 유전자 합성을 통해 진단클론을 제작하였다. 각 클론의 합성유전자를 기반으로 7종의 딸기바이러스 프라이머 세트를 설계하고 편리한 진단법 수행을 위해 동일한 PCR 조건을 설정하였다.

• 한국가시화정보학회지
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• 제14권1호
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• pp.57-61
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• 2016

We report a finding of fast(exceeding 2,000 K/s) heating of polydimethylsiloxane(PDMS), one of the most commonly-used microchannel materials, under cyclic loadings at high(~MHz) frequencies. A microheater was created based on the finding. The heating mechanism utilized vibration damping of sound waves, which were generated and precisely manipulated using a conventional surface acoustic wave(SAW) microfluidic system, in PDMS. The penetration depths were measured to range from $210{\mu}m$ to $1290{\mu}m$, enough to cover most microchannel heights in microfluidic systems. The energy conversion efficiency was SAW frequency-dependent and measured to be the highest at around 30 MHz. Independent actuation of each interdigital transducer(IDT) enabled independent manipulation of SAWs, permitting spatiotemporal control of temperature on the microchip. All the advantages of this microheater facilitated a two-step continuous flow polymerase chain reaction(CFPCR) to achieve the billion-fold amplification of a 134 bp DNA amplicon in less than 3 min. In addition, a technique was developed for establishing dynamic free-form temperature gradients(TGs) in PDMS as well as in gases in contact with the PDMS.