• Animal Systematics, Evolution and Diversity
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• 제36권4호
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• pp.336-346
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• 2020

Mitochondrial genome is an important molecule for systematic and evolutionary studies in metazoans. The development of next-generation sequencing (NGS) technique has rapidly increased the number of mitogenome sequences. The process of generating mitochondrial genome based on NGS includes different steps, from DNA preparation, sequencing, assembly, and annotation. Despite the effort to improve sequencing, assembly, and annotation methods of mitogenome, the low quality and/or quantity sequence in the final map can still be generated through the work. Therefore, it is necessary to check and curate mitochondrial genome sequence after annotation for proofreading and feedback. In this study, we introduce the pipeline for sequencing and curation for mitogenome based on NGS. For this purpose, two mitogenome sequences of Dermatobranchus otome were sequenced by Illumina Miseq system with different amount of raw read data. Generated reads were targeted for assembly and annotation with commonly used programs. As abnormal repeat regions present in the mitogenomes after annotation, primers covering these regions were designed and conventional PCR followed by Sanger sequencing were performed to curate the mitogenome sequences. The obtained sequences were used to replace the abnormal region. Following the replacement, each mitochondrial genome was compared with the other as well as the sequences of close species available on the Genbank for confirmation. After curation, two mitogenomes of D. otome showed a typically circular molecule with 14,559 bp in size and contained 13 protein-coding genes, 22 tRNA genes, two rRNA genes. The phylogenetic tree revealed a close relationship between D. otome and Tritonia diomea. The finding of this study indicated the importance of caution and curation for the generation of mitogenome from NGS.

• BMB Reports
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• 제31권5호
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• pp.475-483
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• 1998

Many antibiotics contain partially deoxygenated sugar components that are usually essential for biological activity, affinity, structural stability, and solubility of antibiotics. Gene probes of the biosynthetic genes related with the deoxysugar were obtained from PCR. Primers were designed from the conserved peptide sequences of the known dTDP-D-glucose 4,6-dehydratases, which are the key step enzymes in the biosynthesis of deoxysugar. The primers were applied to amplify parts of dehydratase genes to 27 actinomycetes that produce the metabolites containing deoxysugar as structural constituents. About 180 and 340 bp DNA fragments from all of the actinomycetes were produced by PCR and analyzed by Southern blot and DNA sequencing. The PCR products were used as gene probes to clone the biosynthetic gene clusters for the antibiotic mithramycin, rubradirin, spectinomycin, and elaiophyrin. This method should allow for detecting of the biosynthetic gene clusters of a vast array of secondary metabolites isolated from actinomycetes because of the widespread existence of deoxysugar constituents in secondary metabolites.

• Mycobiology
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• 제29권3호
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• pp.135-141
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• 2001

Differential display of reverse transcription(DDRT)-PCR was conducted to have a profile of the differentially expressed genes during the formation of fruiting body of Lentinus edodes. The lines of L. edodes(ImHyup-1) employed were cultivated in the artificial blocks of sawdust, and the fruiting body was induced from the mycelia or the mass protruded from the brown surface of the sawdust blocks. RNAs were prepared from the four different developmental stages; mycelial, primordial, and stipes and pileus of fruiting body. The fragments of cDNA were synthesized from the combinations of the arbitrary primers and 3' one anchored Oligo-dT primer. Twelve combinations using the primers have been tested, and among them nineteen bands were identified as differentially expressed. Those genes were further analyzed by DNA sequencing and followed by homology search. Characterization of one clone was conducted as a preliminary data and more are under investigation.

• 대한의생명과학회지
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• 제6권1호
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• pp.73-82
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• 2000

양끝이 같은 형태로 이루어진 DNA조각들은 벡터에 두 가지 방향으로 삽입될 수 있다. 기존의 방법으로는 이들의 방향성을 알아내기 위하여 제한효소의 처리 혹은 DNA염기서열 분석법이 수행되어졌는데, 이들은 적절한 제한효소 인식 부위의 부재 혹은 높은 가격과 많은 샘플수 등으로 그 이용범위가 어느 정도 제한되어 있었다. 본 연구에서는, 벡터에 클로닝 된 DNA조각의 방향성을 결정하기 위한 새로운 실험기법과 이에 따르는 구체적인 방법을 기술하고 이의 직접적인 이용을 보고하고 있다. 통상적인 염기서열 분석용 oligonucleotide primer와 중합효소 연쇄반응용 (PCR) primer를 이용한 PCR에 기초한 이 방법은, 여러 후보 클론의 플라스미드 DNA를 주형으로 하여 한 차례의 반응으로, 원하는 방향으로의 DNA조각이 삽입된 클론을 찾아낼 수 있게 한다. 이 실험기법의 용이함과 정확성은 최근에 보고된 바 있는 랫트의 신경 펩타이드인 urocortin의 cDNA를 재조합 발현 벡터상에 클로닝하고 분석하는 것으로 증명할 수 있었다. 이 같은 방법으로 찾아진 유전자 재조합 클론들은 추가적인 실험을 통하여 CHO 세포주에 transfection 되었는데, 이들이 실제로 urocortin을 발현함은 면역효소 측정법으로 검증될 수 있었고, 이를 통하여 최초로 이 40개의 아미노산으로 이루어진 짧은 펩타이드를 진핵 세포상에서 재조합 단백질의 형태로 발현시키는 데 성공하였다.

• Journal of Animal Science and Technology
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• 제49권5호
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• pp.549-558
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• 2007

C4B 및 BAT2는 SLA class III 영역에 존재하며, 최근 들어 사람의 질병과의 연관성이 보고되고 있다. GenBank database로부터 수집된 사람과 마우스의 C4B 및 BAT2의 CDS를 염기정렬하여 상동성이 높은 부분에서 primer를 제작한 후 RT-PCR 및 RACE-PCR을 수행하여 돼지 C4B 및 BAT2 유전자의 CDS 서열을 결정하였다. 염기서열이 결정된 돼지 C4B와 BAT2의 CDS 길이가 각각 5226 bp와 6501 bp로 나타났다. 이들 각각의 CDS 및 아미노산 서열을 사람 및 마우스와 비교한 결과 CDS는 76~87%, 아미노산 서열은 72~90%의 상동성을 보였으며, C4B가 BAT2에 비해 다소 낮게 나타났다. 두 유전자에서 나타나는 cSNP를 분석하기 위해서 exon 영역을 증폭하기 위한 primer를 제작하였으며, 돼지 6품종을 대상으로 direct sequencing을 실시하였다. 그 결과 C4B로부터 4개, BAT2로부터 3개의 cSNP가 확인되었다. 또한 7개의 cSNP 중 C4B의 C4248T를 제외한 6개의 cSNP에 의해서 아미노산 치환이 발생하였다. 동일한 DNA를 사용하여 7개의 cSNP를 대상으로 Multiplex-ARMS 방법을 사용하여 유전자형 분석을 실시한 결과 direct sequencing 결과와 일치하였다. Multiplex-ARMS 방법의 재현성을 재확인하기 위해 무작위로 2개의 DNA 시료를 선택하여 direct sequencing과 Multiplex-ARMS 분석을 실시하여 유전자형이 일치함을 다시 확인하였다. 따라서 본 연구에서 확인된 7개의 cSNP는 SLA class III 지역의 haplotype 분석을 위한 기초 자료로 사용될 수 있으며, Multiplex- ARMS 기법은 이종장기 개발에 필수적인 SLA 전체 영역 내 유전자들의 유전자형 분석을 위한 효율적인 분석방법이라고 사료된다.

• 한국환경농학회지
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• 제39권1호
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• pp.26-35
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• 2020

BACKGROUND: Microbes that govern a unique biochemical process of oxidizing ammonia into dinitrogen gas, such as anaerobic ammonium oxidation (anammox) have been reported to play a pivotal role in agricultural soils and in oceanic environments. However, limited information for anammox bacterial abundance and distribution in the terrestrial habitats has been known. METHODS AND RESULTS: Phylogenetic and next-generation sequencing analyses of bacterial 16S rRNA gene were performed to examine potential anammox bacteria in paddy soils. Through clone libraries constructed by using the anammox bacteria-specific primers, some clones showed sequence similarities with Planctomycetes (87% to 99%) and anammox bacteria (94% to 95%). Microbial community analysis for the paddy soils by using Illumina Miseq sequencing of 16S rRNA gene at phylum level was dominated by unclassified Bacteria at 33.2 ± 7.6%, followed by Chloroflexi at 20.4 ± 2.0% and Acidobacteria at 17.0 ± 6.5%. Planctomycetes that anammox bacteria are belonged to was 1.5% (± 0.3) on average from the two paddy soils. CONCLUSION: We suggest evidence of anammox bacteria in the paddy soil. In addition to the relatively well-known microbial processes for nitrogen-cycle, anammox can be a potential contributor on the cycle in terrestrial environments such as paddy soils.

• Parasites, Hosts and Diseases
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• 제31권3호
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• pp.285-292
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• 1993

간흡충 total RNk에는 많은 량의 185 rRNA가 함유되어 있었지만 285 rRNA는 그 양이 매우 적었다. 약 $8{\;}{\mu\textrm{g}}의{\;}poly{\;}(A)^{+}$ mRNAS부터 합성된 double-stranded CDNA는 대부분이 0.4-4.2 kb 크기이었으며 9.5 kb에 달하는 것도 있었다. 이미 보고되어 있는 tropomyosin의 amino산 서열을 기준하여 5개의 degenerated oligonucleotide (sense primer 2개와 antisense primer 3개)를 합성하였다. TotalcDNA를 template로 하고 sense primer와 antisense primer를 조합하여 실시한 PCR 산물 중에서 580 bp 크기의 특이 유전자가 나타났다. 만손주혈흡충의 tropomyosin CDNA를 탐색자로 써서 Southern hybridization했을 때 이 유전자만이 검출되어서. 이 유전자는 간횹충 tropomyosin (CSTM) CDNA의 일부분일 가능성이 높다고 생각되어 sequencing vector인 POEM-3Zf(-)에 cloning한 다음 염기서열을 결정하였다. nRf 증폭된 CSTM CDNA는 크기가 575 bp이었으며 191개의 predicted amino산 서열은 한 개의 open reading frame을 갖고 있었다 CSTM CDNA의 amino산 서열은 만손주혈흡충 tropomyosln과 86.3%. Trichosoonvk: colhnfornis tropomyosin과 51.1% 의 유사성을 갖고 있었다. 이 CSTM cDNA fragment는 앞으로 간흡충 cDNA library를 screening하여 완전한 CnM CDNA를 cloning하기에 좋은 probe로 쓰일 것으로 예상된다.

• Ocean and Polar Research
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• 제27권2호
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• pp.205-214
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• 2005

From ancient Antarctic glacier ice, we extracted total genomic DNA that was suitable for prokaryotic 16S rDNA gene cloning and sequencing, and bacterial artificial chromosome (BAC) library and end-sequencing. The ice samples were from the Dry Valley region. Age dating by $^{40}Ar/^{39}Ar$ analysis on the volcanic ashes deposited in situ indicated the ice samples are minimum 100,000-300,000 yr (sample DLE) and 8 million years (sample EME) old. Further assay proved the ice survived freeze-thaw cycles or other re-working processes. EME, which was from a small lobe of the basal Taylor glacier, is the oldest known ice on Earth. Microorganisms, preserved frozen in glacier ice and isolated from the rest of the world over a geological time scale, can provide valuable data or insight for the diversity, distribution, survival strategy, and evolutionary relationships to the extant relatives. From the 16S gene cloning study, we detected no PCR amplicons with Archaea-specific primers, however we found many phylotypes belonging to Bacteria divisions, such as Actinobacteria, Acidobacteria, Proteobacteria $({\alpha},\;{\beta},\;and\;{\gamma})$, Firmicutes, and Cytophaga-Flavobacterium-Bacteroid$. BAC cloning and sequencing revealed protein codings highly identical to phenylacetic acid degradation protein paaA, chromosome segregation ATPases, or cold shock protein B of present day bacteria. Throughput sequencing of the BAC clones is underway. Viable and culturable cells were recovered from the DLE sample, and characterized by their 16S rDNA sequences. Further investigation on the survivorship and functional genes from the past should help unveil the evolution of life on Earth, or elsewhere, if any.

• The Plant Pathology Journal
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• 제32권5호
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• pp.441-451
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• 2016

Deep sequencing has generated 52 contigs derived from five viruses; Apple chlorotic leaf spot virus (ACLSV), Apple stem grooving virus (ASGV), Apple stem pitting virus (ASPV), Apple green crinkle associated virus (AGCaV), and Apricot latent virus (ApLV) were identified from eight apple samples showing small leaves and/or growth retardation. Nucleotide (nt) sequence identity of the assembled contigs was from 68% to 99% compared to the reference sequences of the five respective viral genomes. Sequences of ASPV and ASGV were the most abundantly represented by the 52 contigs assembled. The presence of the five viruses in the samples was confirmed by RT-PCR using specific primers based on the sequences of each assembled contig. All five viruses were detected in three of the samples, whereas all samples had mixed infections with at least two viruses. The most frequently detected virus was ASPV, followed by ASGV, ApLV, ACLSV, and AGCaV which were withal found in mixed infections in the tested samples. AGCaV was identified in assembled contigs ID 1012480 and 93549, which showed 82% and 78% nt sequence identity with ORF1 of AGCaV isolate Aurora-1. ApLV was identified in three assembled contigs, ID 65587, 1802365, and 116777, which showed 77%, 78%, and 76% nt sequence identity respectively with ORF1 of ApLV isolate LA2. Deep sequencing assay was shown to be a valuable and powerful tool for detection and identification of known and unknown virome in infected apple trees, here identifying ApLV and AGCaV in commercial orchards in Korea for the first time.

• 대한본초학회지
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• 제28권6호
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• pp.9-14
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• 2013

Objectives : Cnidii Fructus is prescribed as the fruit of Cnidium monnieri (L.) Cusson or Torilis japonica (Houtt.) DC. in Korea pharmacopoeia. Although there are differences in the composition of useful components, two species have been used without distinction. In order to discriminate them, DNA sequencing and taste pattern analysis were used in this study. Methods : Primers ITS 1 and ITS 4 were used to amplify the intergenic transcribed spacer(ITS) region of nuclear ribosomal DNA from seven T. japonica and six C. monnieri samples. Taste pattern of samples were measured by using taste-sensing system SA402B equipped with five foodstuff sensors(CT0, C00, AAE, CA0, and AE1). The five initial taste(sourness, bitterness, astringency, umami, and saltiness) and three aftertaste(aftertaste of bitterness, astringency, and umami) of two species were compared. Results : According to the results of ITS region sequence analysis, two species showed 94 base pairs differences. The similarity of two sequences was 85%. From the taste pattern analysis, sourness, bitterness, aftertaste of bitterness(aftertaste-B), and umami showed a different pattern. Especially, bitterness and aftertaste-B of C. monnieri were significantly higher than T. japonica. In addition, two species were shown to have two markedly different clustering by these two flavors. Conclusion : T. japonica and C. monnieri were effectively discriminated using DNA sequencing and taste pattern analysis. These methods can be used to identify the origin of traditional medicine in order to maintain therapeutic efficacy.