• 분석과학
• /
• 제31권2호
• /
• pp.65-70
• /
• 2018

이 연구에서는 대규모 병렬형 염기서열 분석 (massively parallel sequencing)에 적용할 샷건 라이브러리 (shotgun library)를 성공적으로 제작하기 위해 두 가지 유형의 고대 DNA (ancient DNA, aDNA) 분리 방법을 비교하였다. 헝가리 선사 시대 늑골 뼈 시료로 실리카 현탁액을 이용한 추출법과 Amicon Ultracel-15 10K 초원심 분리 장치(Millipore)를 이용한 추출법을 비교하였다. 약 150 mg의 뼛가루에서 각각의 방법으로 3 회 반복 추출한 후 이중 가닥 DNA (double stranded DNA, ds DNA)의 양을 측정하였다. 초원심분리 농축 방법은 실리카 현탁액을 사용하는 것보다 더 빠르고, 더 쉬운 공정이며 약 11 배 높은 DNA 회수율을 나타냈다. 또한 초원심 분리 장치로 획득한 DNA 주형은 실리카 현탁액으로 획득한 것보다 샷건 라이브러리가 훨씬 성공적으로 만들어졌다. 두 종류의 aDNA 추출 방법을 비교한 본 연구는 Amicon 장치를 사용하는 분리법이 시간의 절약, 단순한 프로세스 및 높은 효율 등의 장점을 지니고 있음을 보여주었다.

• Genomics & Informatics
• /
• 제2권4호
• /
• pp.174-179
• /
• 2004

We developed various plasmid cloning vectors that are useful in the construction of genomic and shotgun libraries. Two medium copy vectors, pCUGlblu21 (pCb21) and pAGlblu21 (pAb21), which are resistant to kanamycin ($Km^R$) and chloramphenicol ($Cam^R$), respectively, are useful for cloning DNA inserts ranging from 5kb to 15kb. Two high copy vectors, pCUGlblu31 (pCb31) and pAGlblu31 (pAb31), containing $Km^R$ and $Cam^R$, respectively, are useful for DNA inserts less than 5kb. These vectors are well adapted for large-scale genome sequencing projects by providing choice of copy number and selectable marker. The small vector size is another advantage of these vectors. All vectors contain lacZa including multicloning sites that originated from pBluscriptllsk- for easy cloning and sequencing. Two medium copy vectors contain unique and rare cutting Swal (ATTTAAAT) restriction enzyme sites for easy determination of insert size. We developed two combined vectors, pC21A31 and pC31A21, which are combinations of (pCb21 + pAb31) and (pCb31 + pAb21), respectively. These two vectors provide four choices of vectors such as $Km^R$ and medium, $Cam^R$ and high, $Cam^R$ and medium, and $Km^R$ and high copy vectors by restriction enzyme cutting, dephosphorylation, and gel purification. These vectors were successfully applied to high throughput shotgun sequencing of rice, tomato, and brassica BAC clones. With an example of extremely biased hydro sheared 3 kb shotgun library of a tomato BAC clone, which is originated from cytogenetically defined peri-centromeric region, we suggest the utility of an additional 10 kb library for sequence assembly of the difficult-to-assemble BAC clone.

• 한국자원식물학회지
• /
• 제20권6호
• /
• pp.491-498
• /
• 2007

It has been hypothesized that efficient exclusion of methylated retrotransposons and repeated DNA region is one of the rapid and cost-effective approaches for comprehensive gene discovery in large genome size of maize. Three kinds of methylation-sensitive restriction enzymes, HapII, MspI and McrBC, were used to identify the restriction frequency of cytosine methylation sites in maize genome. Roughly 60% of total maize genomic DNA was restricted less than 500bp by McrBC, and the most of restricted small size fraction was composed retrotransposon. In order to validate the efficient construction of gene-rich shotgun library, we compare two gene-rich methyl-filtration shotgun libraries using in vivo and in vitro methyl-filtration system. The size selected DNA fraction by Sau3A-McrBC enzyme treated was very stable and has not appeared modification in E. coli, but most insert DNA size of partially digested with Sau3A were decrease less than 500bp by bacterial methylation-modification system. In compare of retroelements portion, A 44.6% of the sequences were retroelement in unmethyl-filtered library, and the most of them was Copia type, such as Prem, Opie and Ji. The portion of retroelement was drastically decreased to 25% and 20% by in vivo and in vitro filtration system, respectively.

• Journal of Microbiology and Biotechnology
• /
• 제21권2호
• /
• pp.197-203
• /
• 2011

Lactobacilli are normal constituents of the intestinal microbiota, and some strains show the capacity to bind to extracellular matrix proteins and components of the mucosal layer, which represents an adaptation to persist in this niche. A shotgun phage-display library of Lactobacillus casei BL23 was constructed and screened for peptides able to bind to fibronectin and collagen. Clones showing binding to these proteins were isolated, which encoded overlapping fragments of a putative transcriptional regulator (LCABL_29260), a hypothetical protein exclusively found in the L. casei/rhamnosus group (LCABL_01820), and a putative phage-related endolysin (LCABL_13470). The construction of different glutathione S-transferase (GST) fusions confirmed the binding activity and demonstrated that the three identified proteins could interact with fibronectin, fibrinogen, and collagen. The results illustrate the utility of phage display for the isolation of putative adhesins in lactobacilli. However, it remains to be determined whether the primary function of these proteins actually is adhesion to mucosal surfaces.

• Journal of Microbiology and Biotechnology
• /
• 제22권4호
• /
• pp.462-469
• /
• 2012

A metagenomic fosmid library was constructed from genomic DNA isolated from the microbial community residing in hindguts of a wood-feeding higher termite (Microcerotermes sp.) collected in Thailand. The library was screened for clones expressing lignocellulolytic activities. Fourteen independent active clones (2 cellulases and 12 xylanases) were obtained by functional screening at pH 10.0. Analysis of shotgun-cloning and pyrosequencing data revealed six ORFs, which shared less than 59% identity and 73% similarity of their amino acid sequences with known cellulases and xylanases. Conserved domain analysis of these ORFs revealed a cellulase belonging to the glycoside hydrolase family 5, whereas the other five xylanases showed significant identity to diverse families including families 8, 10, and 11. Interestingly, one fosmid clone was isolated carrying three contiguous xylanase genes that may comprise a xylanosome operon. The enzymes with the highest activities at alkaline pH from the initial activity screening were characterized biochemically. These enzymes showed a broad range of enzyme activities from pH 5.0 to 10.0, with pH optimal of 8.0 retaining more than 70% of their respective activities at pH 9.0. The optimal temperatures of these enzymes ranged from $50^{\circ}C$ to $55^{\circ}C$. This study provides evidence for the diversity and function of lignocellulose-degrading enzymes in the termite gut microbial community, which could be of potential use for industrial processes such as pulp biobleaching and denim biostoning.

• 한국임상수의학회지
• /
• 제36권4호
• /
• pp.190-195
• /
• 2019

We aimed to identify genomic variations as well as the marker genes related with chronic mitral valve disease (CMVD) in Canis lupus familiaris using whole genome resequencing, which provides valuable resources for further study. Two ten-year old female Canis lupus familiaris English cocker spaniels were used for this study, one control and one who had been diagnosed as CMVD. For the whole genome resequencing, muscles from the left ventricular wall were collected from each dog. With the HiSeq DNA Shotgun library and $HiSeq^{TM}$ 2000 platform, whole genome resequencing was performed. From the results, we identified 5 million and 6 million variants in gene expression in the control and CMVD-diagnosed subject, respectively. We then selected the top 1,000 genes from the SNP, INS, and DEL mutation and 675 genes among them were overlapped for every mutation between the control and CMVD-diagnosed patient. Interestingly, in both groups, the intron variant (91.16 and 91.18%) and upstream variant (3.10 and 3.08%) are most highly related. Among the overlapped 675 genes, gene ontology for intracellular signal transduction is highly counted in INS, and DEL, and SNPs (35, 33, 31, respectively). In this study, we found that the COL and CDH gene families could be key molecules in identifying the difference in gene expression between control and CMVD-diagnosed dogs. We believe further studies will prove the importance of variants in key molecule expression and that these data will serve as a valuable foundation stone the study of canine CMVD.