• Title/Summary/Keyword: Transcription Method

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Compiling Multicopy Single-Stranded DNA Sequences from Bacterial Genome Sequences

  • Yoo, Wonseok;Lim, Dongbin;Kim, Sangsoo
    • Genomics & Informatics
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    • v.14 no.1
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    • pp.29-33
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    • 2016
  • A retron is a bacterial retroelement that encodes an RNA gene and a reverse transcriptase (RT). The former, once transcribed, works as a template primer for reverse transcription by the latter. The resulting DNA is covalently linked to the upstream part of the RNA; this chimera is called multicopy single-stranded DNA (msDNA), which is extrachromosomal DNA found in many bacterial species. Based on the conserved features in the eight known msDNA sequences, we developed a detection method and applied it to scan National Center for Biotechnology Information (NCBI) RefSeq bacterial genome sequences. Among 16,844 bacterial sequences possessing a retron-type RT domain, we identified 48 unique types of msDNA. Currently, the biological role of msDNA is not well understood. Our work will be a useful tool in studying the distribution, evolution, and physiological role of msDNA.

TALENs Construction: Slowly but Surely

  • Hegazy, Wael Abdel Halim;Youns, Mahmoud
    • Asian Pacific Journal of Cancer Prevention
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    • v.17 no.7
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    • pp.3329-3334
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    • 2016
  • Cancer is thought to be a direct result of transcriptional misregulation. Broad analysis of transcriptional regulatory elements in healthy and cancer cells is needed to understand cancer development. Nucleases regulatory domains are recruited to bind and manipulate a specific genomic locus with high efficacy and specificity. TALENs (transcription activator-like effector nuclease) fused to endonuclease FokI have been used widely to target specific sequences to edit several genes in healthy and cancer cells. This approach is promising to target specific cancer genes and for this purpose it is needed to pack such TALENs into viral vectors. There are some considerations which control the success of this approach, targeting appropriate sequences with efficient construction of TALENs being crucial factors. We face some obstacles in construction of TALENs; in this study we made a modification to the method of Cermk et al 2011 and added one step to make it easier and increase the availability of constructs.

Nucleotide Sequence Analysis of Movement Protein Gene from Tobacco Mosaic Virus Korean Pepper (TMV-KP) Strain (담배 모자이크 바이러스 한국고추계통에서 분리한 이동 단백질 유전자의 염기서열 분석)

  • 이재열;정동수;장무웅;최장경
    • Korean Journal Plant Pathology
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    • v.11 no.1
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    • pp.87-90
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    • 1995
  • Complementary DNA of the movement protein (MP) gene of tobacco mosaic virus Korean pepper strain (TMV-KP) was synthesized from purified TMV-KP RNA by using the reverse transcription and polymerase chain reaction (PCR) system. The synthesized double stranded cDNA was cloned into the plasmid pUC9 and transformed into Escherichia coli JM110. The movement protein gene of TMV-KP of the selected clones was subjected to sequence analysis by Sanger's dideoxy chain termination method. The complete sequence of viral MP gene from TMV-KP strain was 807 nucleotides long. The nucleotide of MP gene from TMV-KP has thirteen and two nucleotide differences from TMV vulgarae (TMV-OM) and Korean (TMV-K) strains, respectively. Thus, the nucleotide sequence of TMV-KP MP gene showed higher homology of 99% with that of TMV-K MP gene.

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Diagnosis of Potato Leafroll Virus with Digoxigenin-labeled cRNA Probes (Digoxigenin으로 표지된 cRNA 프로브를 이용한 감자잎말림바이러스(PLRV)의 짐단)

  • 서효원;함영일;오승은;신관용;최장경
    • Korean Journal Plant Pathology
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    • v.14 no.6
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    • pp.636-641
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    • 1998
  • Digoxigenin (DIG) was used to prepare nucleic acid probe for the detection of RNA of potato leafroll virus (PLRV) in the potato leaf extracts. The 0.6 kb coat protein (CP) gene cDNA of PLRV in plasmid pSPT 18 vector was labeled with digoxigenin by in vitro run-off transcription and then used for cRNA probe. In the several buffers tested for increase the total RNA extraction efficiency AMES buffer was the most suitable for this detection method. The RNA extracts from potato leaves shown symptoms of PLRV were dot blotted onto nylon membrane and hybridized with labeled RNA probes. After hybridization, labeled RNA bound to PLRV RNA on membrane was detected with anti-digoxigenin alkaline phosphatase. 5-bromo-4-chloro-3-indolyl-phosphate/nitroblue tetrazolium (NBT) salt and CSPD were used as substrate for colorimetric and film exposure detection, respectively. These detection methods were very sensitive allowing for detection of 1/32 diluted total RNA extract from 100 mg leaf tissue.

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Reduction of muscle cyclooxygenase-2 with transcutaneous electrical nerve stimulation and cold therapy in rats of carrageenan-induced inflammatory muscle pain (Carrageenan으로 유도된 염증성 근통증 흰쥐 모델에서 경피신경전기자극과 냉치료에 의한 비복근의 cyclooxygenase-2의 감소)

  • Paek, Yun-Woong;Chae, Yun-Won
    • Journal of Korean Physical Therapy Science
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    • v.9 no.1
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    • pp.89-94
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    • 2002
  • Prostaglandins are generated through two isoforms of the enzyme cyclooxygenase, constitutively expressed cyclooxygenase(COX)-1 and COX-2, which is induced at sites of inflammation. Inhibition of COX-2 is desirable as this may avoid side effects seen with NSAIDs. We examined the effects of transcutaneous electrical nerve stimulation and cold therapy on the levels of muscle cycloooxygenase-2 mRNA in rats of carrageenan-induced inflammatory. The method of behavioral assessment were paw withdrawal latency(PWL) and tail flick test(TFT). The COX-2 mRNA levels were quantified by reverse transcription-polymerase chain reaction (RT-PCR). Following the transcutaneous electrical nerve stimulation and cold therapy, PWL and TFT were increased and COX-2 mRNA expression in gastrocnemius muscles were decreased. These results suggest that a transcutaneous electrical nerve stimulation and cold therapy were good therapy for a muscle pain.

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Innovations in Micro Metal Injection Molding Process by Lost Form Technology

  • Nishiyabu, Kazuaki;Kanoko, Yasuhiro;Tanaka, Shigeo
    • Proceedings of the Korean Powder Metallurgy Institute Conference
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    • 2006.09a
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    • pp.43-44
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    • 2006
  • The production method of micro sacrificial plastic mold insert metal injection molding, namely ${\mu}-SPiMIM$ process has been proposed to solve specific problems involving the miniaturization of MIM. Two types of sacrificial plastic molds (SP-mold) with fine structures were used: 1) PMMA resist, 2) PMMA mold injected into Ni-electroform, which is a typical LIGA (${\underline{L}}ithographie-{\underline{G}}alvanoformung-{\underline{A}}bformung$) process. Stainless steel 316L feedstock was injection-molded into the SP-molds with multi-pillar structures. This study focused on the effects of metal particle size and processing conditions on the shrinkage, transcription and surface roughness of sintered parts.

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A Study on Earth-Moon Transfer Orbit Design

  • No, Tae-Soo;Lee, Ji-Marn;Jeon, Gyeong-Eon;Lee, Dae-Ro;Kim, Ghang-Ho
    • International Journal of Aeronautical and Space Sciences
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    • v.13 no.1
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    • pp.106-116
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    • 2012
  • Optimal transfer trajectories based on the planar circular restricted three body problem are designed by using mixed impulsive and continuous thrust. Continuous and dynamic trajectory optimization is reformulated in the form of discrete optimization problem. This is done by the method of direct transcription and collocation. It is then solved by using nonlinear programming software. Two very different transfer trajectories can be obtained by the different combinations of the design parameters. Furthermore, it was found out that all designed trajectories permit a ballistic capture by the Moon's gravity. Finally, the required thrust profiles are presented and they are analyzed in detail.

Diagnosis of porcine reproductive and respiratory syndrome (PRRS) and its serological survey using the reverse transcription and polymerase chain reaction (RT-PCR) and ELISA (RT-PCR과 ELISA를 이용한 PRRS 진단 및 항체가 조사)

  • Chu Keum-Suk;Han Keu-Sam;Han Jae-Cheol;Song Hee-Jong
    • Korean Journal of Veterinary Service
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    • v.27 no.3
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    • pp.273-280
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    • 2004
  • The studies were performed for the PRRS antigen and antibody detection from breeding farms, artificial insemination(AI) center and growing farms in Jeonbuk province. 1. Specific PRRS primers were successfully amplified ORF6 617bp and ORF7 448 bp on agarose gel. 2. RT-PCR method has been establish by commercial kit and the thermal cycler program consisted of 30 cycles: $95^{\circ}C$ for 30 sec, $45^{\circ}C$ for 30 sec, and $72^{\circ}C$ for 45 sec. 3. The results of PRRS antibody test by ELISA method in AI centers were $6.6\%,\;53.3\%$ and breeding farms $65\%,\;65\%\;and\;38.7\%$, respectively. The serological positive of the antibody in gilt higher than sow. 4. The sero-positive of the PRRS antibody showed average $21\%$ in domestic farms, $56.2\%$ in breeding farms, and $29.9\%$ in AI center.

Reverse Transcription and Amplification of Halobacterial gvp Genes with Polymerase Chain Reaction Method (Polymerase Chain Reaction 방법에 의한 Halobacteria gvp 유전자의 역전사 및 증폭)

  • 윤병수;이상섭
    • Korean Journal of Microbiology
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    • v.30 no.6
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    • pp.456-459
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    • 1992
  • The genes of Halohacteria. gvpD and gvpE. take part in formation of gas vesicle. These mRNA cause a lot of experimental prohlems due to its eharacteristic instahility in the analysis of transcripts. This study allowed easy cloning and sequencing of RNA hy substituting a stable complementary DNA for the mRNA of the genes for an analysis. The weak 111 RNA was reverse transcribed to DNA using reverse transcriptase. and was amplified using PCR method. The transcripts confirmed in this ~,tudy have not heen round in the northern hybridization covering almost all ranges of ORF of the gene. gvpD.

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Rapid Screening of Apple mosaic virus in Cultivated Apples by RT-PCR

  • Ryu, Ki-Hyun;Park, Sun-Hee
    • The Plant Pathology Journal
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    • v.19 no.3
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    • pp.159-161
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    • 2003
  • The coat protein (CP) gene of Apple mosaic virus (ApMV), a member of the genus Ilarvirus, was selected for the design of virus-specific primers for amplification and molecular detection of the virus in cultivated apple. A combined assay of reverse transcription and polymerase chain reaction (RT-PCR) was performed with a single pair of ApMV-specific primers and crude nucleic acid extracts from virus-infected apple for rapid detection of the virus. The PCR product was verified by restriction mapping analysis and by sequence determination. The lowest concentration of template viral RNA required for detection was 100 fg. This indicates that the RT-PCR for detection of the virus is a 10$^3$times more sensitive, reproducible and time-saving method than the enzyme-linked immunosorbent assay. The specificity of the primers was verified using other unrelated viral RNAs. No PCR product was observed when Cucumber mosaic virus (Cucumovirus) or a crude extract of healthy apple was used as a template in RT-PCR with the same primers. The PCR product (669 bp) of the CP gene of the virus was cloned into the plasmid vector and result-ant recombinant (pAPCP1) was selected for molecule of apple transformation to breed virus-resistant transgenic apple plants as the next step. This method can be useful for early stage screening of in vitro plantlet and genetic resources of resistant cultivar of apple plants.