• Title/Summary/Keyword: Insert size

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Construction of cDNA Library for Using Virus-induced Gene Silencing (VIGS) Vector with the Sweetpotato Whitefly, Bemisia tabaci(Hemiptera: Aleyrodidae) (담배가루이(Bemisia tabaci, Aleyrodidae, Hemiptera)에서 Virus-induced Gene Silencing (VIGS) Vector를 이용하기 위한 cDNA Library 제작)

  • Ko, Na Yeon;Lim, Hyoun Sub;Yu, Yong Man;Youn, Young Nam
    • Korean journal of applied entomology
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    • v.54 no.2
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    • pp.91-97
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    • 2015
  • The sweetpotato whitefly, Bemisia tabaci, is the major insect pest that transmitted over 100 plant viruses including tomato yellow leaf curl virus (TYLCV) of tomato plant as virus vector in the world. In this study, cDNA library of whitefly was constructed using Gateway system for selecting target gene in order to control of B. tabaci using virus-induced gene silencing (VIGS) vector with RNAi. First of all, when using oligo d(T) rimer, the calculated titer of cDNA library was confirmed with $1.4{\times}10^4$ clones and average insert sizes was confirmed with 1 kb. However, insert size was very big for construction of cDNA. Otherwise, when using attB-N25 random primer and sonication for 6 sec, the calculated titer of cDNA library was confirmed with $1.04{\times}10^5$ clones. But mostly insert band wasn't identified on the electrophoresis, because it seemed that insert size is too small (${\leq}100bp$), also the size of identified insert was somewhat big. Finally, when using oligo d(T) primer and sonication for 1 sec, cDNA insert of whitefly was appropriated for VIGS with 300-600 bp. However, cDNA sequence included a poly A and titer was very low to $5.2{\times}10^2$ clones. It was supposed that heat shock transformation was used instead of electro-transformation. It is considered that when constructing cDNA library for using VIGS vector, (1) random primer should be used for First strand cDNA synthesis in order to remove poly A and (2) sonication for 1 sec should be performed in order to get appropriated insert size and (3) electro-transformation should be performed in order to improve transformation efficiency.

Performance estimation depending on the insert size of conical picks by linear cutting test (선형절삭시험에 의한 코니컬커터의 삽입재 크기에 따른 절삭성능 평가)

  • Choi, Soon-Wook;Kang, Tae-Ho;Chang, Soo-Ho;Lee, Cheol-Ho;Lee, Gyu-Phil;Kim, Chang-Yong
    • Journal of Korean Tunnelling and Underground Space Association
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    • v.18 no.2
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    • pp.221-233
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    • 2016
  • In order to estimate the performance of a conical cutter depending on the insert size, this study measured forces acting on conical cutters with different cutter spacings, penetration depths and skew angles using slim and heavy conical cutters. When cutter spacings ranged from 12 to 27 mm, the deviations of mean cutter forces with cutter depths appeared smaller compared to other cutter spacings. When skew angle is $0^{\circ}$, the optimal S/d ratio was obtained in the range of 4 to 4.5 for which specific energy of cutting was minimized. It were usually found in the range of 1 to 5. However, when skew angle is $6^{\circ}$, the optimal S/d ratio was obtained in the range of 1 to 3. The simple comparison results shows that the performance of slim cutter was superior to that of heavy cutter, but the use of heavy cutter can be effective, considering the cutter consumption and cutter damage when the strength of the ground is high enough.

Instability of pneumococcus library in pHC79 and pAcyc184

  • Rhee, Dong-Kwon
    • Archives of Pharmacal Research
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    • v.18 no.1
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    • pp.31-37
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    • 1995
  • S. pneumoniae (pneumococcus) gene cloning and library construction in E. coli multicopy plasmid has been hampered, in part, by instability problems. In this study, stability of pneumococcus gene library in cosmid vector and pACYC184 was examined. Pneumococcus library in the cosmid vector pHC79 was extermely unstable that most of the recobinant clones were degenerated rapidly. Only 2 out 849 clones were stable and had appropriate insert size. Pneumococcus library in pACYC184 was also so unstable that the pneumococcal inserg and/or part of the vector were deleted. However, the instability problems seemed to be resolved when transcription teminator plasmid was employed for pneumococcus library construction.

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Korean BAC Library Construction and Characterization of HLA-DRA, HLA-DRB3

  • Park, Mi-Hyun;Lee, Hye-Ja;Bok, Jeong;Kim, Cheol-Hwan;Hong, Seong-Tshool;Park, Chan;Kimm, Ku-Chan;Oh, Berm-Seok;Lee, Jong-Young
    • BMB Reports
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    • v.39 no.4
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    • pp.418-425
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    • 2006
  • A human bacterial artificial chromosome (BAC) library was constructed with high molecular weight DNA extracted from the blood of a male Korean. This Korean BAC library contains 100,224 clones of insert size ranging from 70 to 150 kb, with an average size of 86 kb, corresponding to a 2.9-fold redundancy of the genome. The average insert size was determined from 288 randomly selected BAC clones that were well distributed among all the chromosomes. We developed a pooling system and three-step PCR screen for the Korean BAC library to isolate desired BAC clones, and we confirmed its utility using primer pairs designed for one of the clones. The Korean BAC library and screening pools will allow PCR-based screening of the Korean genome for any gene of interest. We also determined the allele types of HLA-DRA and HLA-DRB3 of clone KB55453, located in the HLA class II region on chromosome 6p21.3. The HLA-DRA and DRB3 genes in this clone were identified as the DRA*010202 and DRB3*01010201 types, respectively. The haplotype found in this library will provide useful information in future human disease studies.

Durability evaluation depending on the insert size of conical Picks by the field test (삽입재 크기에 따른 코니컬 커터의 현장 내구성 평가 연구)

  • Choi, Soon-Wook
    • Journal of Korean Tunnelling and Underground Space Association
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    • v.21 no.1
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    • pp.49-59
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    • 2019
  • In this study, the durability of conical pick cutter was compared and analyzed by pre- and post-test visual inspection, measurement of weight loss and wear volume through field test on two types of conical pick cutters applied to rotary drum cutter. In the visual inspection, it was found that only 9 inserts were lost in the slim type conical pick cutter. This result show that the thickness of the head cover surrounding a insert was important to maintain the insert during excavation. The weight loss and wear volume of the heavy type conical pick cutter were less than half that of the slim type. From these results, it can be confirmed that heavy type is more useful than slim type in hard rock. It should be noted that, when determining the wear loss of the conical pick cutter, the mutual comparison of the weight measurement and the wear volume measurement results may be different due to the unit weight of the material and the spalling caused by excavation.

Enhanced Transformation Efficiency of an Anticoagulant Hirudin Gene into Saccharomyces cerevisiae by a Double ${\delta}-Sequence$

  • Kim, Myoung-Dong;Yoo, Young-Je;Rhee, Sang-Ki;Seo, JIn-Ho
    • Journal of Microbiology and Biotechnology
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    • v.11 no.1
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    • pp.61-64
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    • 2001
  • Delta-integration vectors were constructed for the purpose of achieving homologous integration of the hirudin expression cassette into the chromosome of Saccharomyces cerevisiae. A double $\delta$ system truncated with the unnecessary bacterial genes, and consequently having a reduced insert size for integration, showed a four-fold increase in transformation efficiency at given DNA concentrations, and as a result, the constructed recombinant yeast strain had a 1.3-fold enhancement in hirudin expression level compared with a single $\delta$ system.

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Construction of Chromosome-Specific BAC Libraries from the Filamentous Ascomycete Ashbya gossypii

  • Choi Sang-Dun
    • Genomics & Informatics
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    • v.4 no.2
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    • pp.80-86
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    • 2006
  • It is clear that the construction of large insert DNA libraries is important for map-based gene cloning, the assembly of physical maps, and simple screening for specific genomic sequences. The bacterial artificial chromosome (BAC) system is likely to be an important tool for map-based cloning of genes since BAC libraries can be constructed simply and analyzed more efficiently than yeast artificial chromosome (YAC) libraries. BACs have significantly expanded the size of fragments from eukaryotic genomes that can be cloned in Escherichia coli as plasmid molecules. To facilitate the isolation of molecular-biologically important genes in Ashbya gossypii, we constructed Ashbya chromosome-specific BAC libraries using pBeloBAC11 and pBACwich vectors with an average insert size of 100 kb, which is equivalent to 19.8X genomic coverage. pBACwich was developed to streamline map-based cloning by providing a tool to integrate large DNA fragments into specific sites in chromosomes. These chromosome-specific libraries have provided a useful tool for the further characterization of the Ashbya genome including positional cloning and genome sequencing.

Comparison of Functional Gene Annotation of Toxascaris leonina and Toxocara canis using CLC Genomics Workbench

  • Kim, Ki Uk;Park, Sang Kyun;Kang, Shin Ae;Park, Mi Kyung;Cho, Min Kyoung;Jung, Ho-Jin;Kim, Kyung-Yun;Yu, Hak Sun
    • The Korean Journal of Parasitology
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    • v.51 no.5
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    • pp.525-530
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    • 2013
  • The ascarids, Toxocara canis and Toxascaris leonina, are probably the most common gastrointestinal helminths encountered in dogs. In order to understand biological differences of 2 ascarids, we analyzed gene expression profiles of female adults of T. canis and T. leonina using CLC Genomics Workbench, and the results were compared with those of free-living nematode Caenorhabditis elegans. A total of 2,880 and 7,949 ESTs were collected from T. leonina and T. canis, respectively. The length of ESTs ranged from 106 to 4,637 bp with an average insert size of 820 bp. Overall, our results showed that most functional gene annotations of 2 ascarids were quite similar to each other in 3 major categories, i.e., cellular component, biological process, and molecular function. Although some different transcript expression categories were found, the distance was short and it was not enough to explain their different lifestyles. However, we found distinguished transcript differences between ascarid parasites and free-living nematodes. Understanding evolutionary genetic changes might be helpful for studies of the lifestyle and evolution of parasites.

Construction of a Bacterial Artificial Chromosome Library Containing Large BamHI Genomic Fragments from Medicago truncatula and Identification of Clones Linked to Hypernodulating Genes

  • Park So-Yeon;Nam Young-Woo
    • Journal of Microbiology and Biotechnology
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    • v.16 no.2
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    • pp.256-263
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    • 2006
  • In the model legume Medicago truncatula, two mutants, sickle and sunn, exhibit morphologically and genetically distinct hypernodulation phenotypes. However, efforts to isolate the single recessive and single semidominant genes for sickle and sunn, respectively, by map-based cloning have so far been unsuccessful, partly due to the absence of clones that enable walks from linked marker positions. To help resolve these difficulties, a new bacterial artificial chromosome (BAC) library was constructed using BamHI-digested genomic fragments. A total of 23,808 clones were collected from ligation mixtures prepared with double-size-selected high-molecular-weight DNA. The average insert size was 116 kb based on an analysis of 88 randomly selected clones using NotI digestion and pulsed-field gel electrophoresis. About 18.5% of the library clones lacked inserts. The frequency of the BAC clones carrying chloroplast or mitochondrial DNA was 0.98% and 0.03%, respectively. The library represented approximately 4.9 haploid M. truncatula genomes. Hybridization of the BAC clone filters with a $C_{0}t-l$ DNA probe revealed that approximately 37% of the clones likely carried repetitive sequence-enriched DNA. An ordered array of pooled BAC DNA was screened by polymerase chain reactions using 13 sequence-characterized molecular markers that belonged to the eight linkage groups. Except for two markers, one to five positive BAC clones were obtained per marker. Accordingly, the sickle- and sunn-linked BAC clones identified herein will be useful for the isolation of these biotechnologically important genes. The new library will also provide clones that fill the gaps between preexisting BAC contigs, facilitating the physical mapping and genome sequencing of M. truncatula.

Genes of Wild Rice (Oryza grandiglumis) Induced by Wounding and Yeast Extract (상처와 효모추출물 처리조건에서 유발되는 야생벼 유전자 스크린)

  • Shin, Sang-Hyun;Im, Hyun-Hee;Lee, Jai-Heon;Kim, Doh-Hoon;Chung, Won-Bok;Kang, Kyung-Ho;Cho, Sung-Ki;Shin, Jeong-Sheop;Chung, Young-Soo
    • Journal of Life Science
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    • v.14 no.4
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    • pp.650-656
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    • 2004
  • Oryza grandiglumis (CCDD, 2n=48), one of the wild rice species, has been known to possess fungal-,bacterial-, and insect-resistance against sheath blight, rice blast, bacterial leaf blight and brown plant hopper (Nilaparvata lugens). To rapidly isolate differentially expressed genes responding to fungal and wounding stress, wounding and yeast extract were treated to O. grandiglumis for 24 hrs. Suppression subtractive hybridization (SSH) method was used to obtain differentially expressed genes from yeast extract and wounding treated plants. Seven hundreds and seventy six clones were obtained by subcloning PCR product, and colony array and screening were carried out using radio-isotope labeled cDNA probes prepared from the wounding and yeast extract treated plants. One hundred and fifteen colonies were confirmed as true positive ones. Average insert size of the clones were ranged from 400 bp to 700 bp and all the inserts were sequenced. To decide the identity of those clones, sequences were analyzed by sequence homology via GenBank database. The homology search result showed that 68 clones were matched to the genes with known function; 16 were related to primary metabolism, 5 to plant retrotransposons, 5 to defense related metallothionein-like genes. In addition to that, others were matched to various genes with known function in amino acid synthesis and processing, membrane transport, and signal transduction, so on. In northern blot analysis, induced expressions of ogwfi-161, ogwfi-646, ogwfi-663, and ogwfi-695 by wounding and yeast extract treatments were confirmed. The result indicates that SSH method is very efficient for rapid screening of differentially expressed genes.