• Title/Summary/Keyword: DNA array

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Gene Editing for Major Allergy Genes using Multiplex CRISPR-Cas9 System & Prime Editing in Peanuts (Arachis hypogaea L.)

  • Min-cheol Kim;Tae-Hwan Jun
    • Proceedings of the Korean Society of Crop Science Conference
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    • 2022.10a
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    • pp.200-200
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    • 2022
  • Recently, food-induced allergies have emerged as major global concerns. In the past ten years, it has doubled in western nations, and it has also increased in Asia and Africa. In many cases of food allergy, peanut allergy is prevalent, typically permanent, and frequently life-threatening. Therefore, we utilized gene editing techniques on the three major allergen genes in peanuts, Ara h 1, Ara h 2, and Ara h 3. Using gibson assembly and golden gate assembly, we created two vectors, the gRNA-tRNA array CRISPR-Cas9 system and Prime-editing. Using LBA4404 strain and agrobacterium-mediated transformation, the vectors were transferred to two elite Korean peanut lines. After co-cultivation and tissue culture, we extracted the tissue cultured peanut DNA amplified the hygromycin resistance gene and Cas9 gene in the T-DNA region. The integration of the T-DNA region into the host genome was demonstrated by the presence of a specific band in some samples. There have only been a few reported peanut gene editing studies. So, this study will contribute to peanut allergy and gene editing research.

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Construction of the Full-length cDNA Library and Selection of Diapause-Associated cDNA Clones from Bombyx mori Diapausing Eggs and Diapause-Activated Eggs

  • Hwnag, Jae-Sam;Go, Hyun-Jeong;Goo, Tae-Won;Yun, Eun-Young;Choi, Kwang-Ho;Seong, Su-Il;Kim, Sung-Kuk;Lee, Sang-Mong;Kim, Iksoo;Kim, Nam-Soon;Kang, Seok-Woo
    • International Journal of Industrial Entomology and Biomaterials
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    • v.9 no.1
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    • pp.145-148
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    • 2004
  • As an initial step to define the molecular mechanism of diapause during embryogenesis of the silkworm, Bombyx mori, mRNA transcripts from diapausing eggs and diapause-activated eggs were compared by differential expression using cDNA microarray. Twenty-four individual cDNA clones were identified. Amomg them, ten genes including alcohol dehydrogenase, dead box-l, cytochrome oxidase subunit I and 18 wheeler showed increased expression in the diapause-activated eggs. The rest of fourteen genes showed increased expression in diapausing eggs.

A Fluorescence-based cDNA-AFLP Method for Identification of Differentially Expressed Genes

  • Park, Sook-Young;Jwa, Nam-Soo;Chi, Myoung-Hwan;Lee, Yong-Hwan
    • The Plant Pathology Journal
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    • v.25 no.2
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    • pp.184-188
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    • 2009
  • Identification of differently expressed genes under specific tissues and/or environments provides insights into the nature and underlying mechanisms of cellular processes. Although cDNA-AFLP (Amplified Fragment Length Polymorphism) is a powerful method for analyzing differentially expressed genes, its use has been limited to the requirement of radioactive isotope use and the difficulty of isolating the bands of interest from a gel. Here, we describe a modified method for cDNA-AFLP that uses a fluorescence dye for detection and isolation of bands directly from a small size polyacrylamide gel. This method involves three steps: (i) preparation of cDNA templates, (ii) PCR amplification and differential display, and (iii) identification of differentially expressed genes. To demonstrate its utility and efficiency, differentially expressed genes during vegetative growth and appressorial development of Magnaporthe oryzae were analyzed. This method could be applied to compare gene expression profiles in a diverse array of organisms.

Recyclable single-stranded DNA template for synthesis of siRNAs

  • Ali, Mussa M.;Obregon, Demian;Agrawal, Krishna C.;Mansour, Mahmoud;Abdel-Mageed, Asim B.
    • BMB Reports
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    • v.43 no.11
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    • pp.732-737
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    • 2010
  • RNA interference is a post-transcriptional silencing mechanism triggered by the bioavailability and/or exogenous introduction of double-stranded RNA (dsRNA) into cells. Here we describe a novel method for the synthesis of siRNA in a single vessel. The method employs in vitro transcription and a single-stranded DNA (ssDNA) template and design, which incorporates upon self-annealing, two promoters, two templates, and three loop regions. Using this method of synthesis we generated efficacious siRNAs designed to silence both exogenous and endogenous genes in mammalian cells. Due to its unique design the single-stranded template is easily amenable to adaptation for attachment to surface platforms for synthesis of siRNAs. A siRNA synthesis platform was generated using a 3' end-biotinylated ssDNA template tethered to a streptavidin coated surface that generates stable siRNAs under multiple cycles of production. Together these data demonstrate a unique and robust method for scalable siRNA synthesis with potential application in RNAi-based array systems.

Biodistribution and Genotoxicity of Transferrin-Conjugated Liposomes/DNA Complexes in Mice

  • Lee Sang Mi;Kim Jin-Seok;Oh Yu-Kyoung;Lee Yong-Bok;Sah Hongkee
    • Macromolecular Research
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    • v.13 no.3
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    • pp.218-222
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    • 2005
  • Transferrin-conjugated liposomes ($T_f$-liposomes) were made and formulated with pCMVluc DNA to form a lipoplex. Among the various formulations studied, the $T_f$-liposome: pCMVluc DNA complex at a ratio of 5: 1 (wt/wt) showed the highest transfection efficiency, which was twice that of $Lipofectin^{TM}$ on HeLa cells. The maxi-mum tolerated dose (MTD) of this lipoplex formulation from a single intravenous injection was over 10 mg/kg in healthy ICR mice. The RT-PCR results showed that the highest level of luciferase mRNA was detected in the lungs, followed by the liver, spleen, heart and kidneys, after an intravenous injection into mice. Two weeks after the injection, the levels of luciferase mRNA decreased gradually in the liver, spleen, heart, and kidney, but not in the lungs. The micro-array study showed that the cancer-related genes, including the bcl 6 gene, were highly up-regulated by the treatment with $T_f$-liposome/ pCMVluc DNA complex on HeLa cells, indicating that there were possible interactions between the host chromosomal DNA and the $T_f$-liposome within the cells. The results obtained from this study are expected to be useful for designing a safe and efficient gene delivery system using transferrin-conjugated liposomes.

Comparison of Expression Profiling of Gastric Cancer by O1igonucleotide and cDNA Microarrays (O1igonucleotide Microarray와 cDNA Microarray를 이용한 위암조직의 대단위 유전자 발현 비교)

  • Jung, Kwang-Hwa;Kim, Jung-Kyu;Noh, Ji-Heon;Eun, Jung-Woo;Bae, Hyun-Jin;Lee, Sug-Hyung;Park, Won-Sang;Yoo, Nam-Jin;Lee, Jung-Young;Nam, Suk-Woo
    • YAKHAK HOEJI
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    • v.51 no.3
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    • pp.179-185
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    • 2007
  • Gastric cancer is one of the most common malignancies in Korea, but the predominant molecular event underlying gastric carcinogenesis remain unknown. Recently, DNA microarray technology has enabled the comprehensive analysis of gene expression level, and as such has yielded great insight into the molecular nature of cancer, However, despite the powerful approach of this techniques, the technical artifacts and/or bias in applied array platform limited the liability of resultant tens of thousand data points from microarray experiments. Therefore, we applied two different any platforms, such as olignucleotide microarray and cDNA microarray, to identify gastric cancer related large-scale molecular signature of the same human specimens. When thirty sets of matched human gastric cancer and normal tissues subjected to oligonucleotide microarray, total 623 genes were resulted as differently expressed genes in gastric cancer compared to normal tissues, and 252 genes for cDNA microarray analysis. In addition, forty three outlier genes which reflect the characteristic expression signature of gastric cancer beyond array platform and analytical protocol was recapitulated from two different expression profile. In conclusion, we were able to identify robust large-scale molecular changes in gastric cancer by applying two different platform of DNA microarray, this may facilitate to understand molecular carcinogenesis of gastric cancer.

An Efficient Data Structure to Obtain Range Minima in Constant Time in Constructing Suffix Arrays (접미사 배열 생성 과정에서 구간 최소간 위치를 상수 시간에 찾기 위한 효율적인 자료구조)

  • 박희진
    • Journal of KIISE:Computer Systems and Theory
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    • v.31 no.3_4
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    • pp.145-151
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    • 2004
  • We present an efficient data structure to obtain the range minima in an away in constant time. Recently, suffix ways are extensively used to search DNA sequences fast in bioinformatics. In constructing suffix arrays, solving the range minima problem is necessary When we construct suffix arrays, we should solve the range minima problem not only in a time-efficient way but also in a space-efficient way. The reason is that DNA sequences consist of millions or billions of bases. Until now, the most efficient data structure to find the range minima in an way in constant time is based on the method that converts the range minima problem in an array into the LCA (Lowest Common Ancestor) problem in a Cartesian tree and then converts the LCA problem into the range minima problem in a specific array. This data structure occupies O( n) space and is constructed in O(n) time. However since this data structure includes intermediate data structures required to convert the range minima problem in an array into other problems, it requires large space (=13n) and much time. Our data structure is based on the method that directly solves the range minima problem. Thus, our data structure requires small space (=5n) and less time in practice. As a matter of course, our data structure requires O(n) time and space theoretically.

Identifying Genes Related with Radiation Resistance in Oral Squamous Cell Carcinoma Cell Lines (두경부 편평상피세포암주에서 방사선 내성 관련 유전인자의 확인)

  • Lee, Sei-Young;Seol, Jeong-Hun;Park, Haeng-Ran;Cho, Nam-Hoon;Choi, Yoon-Pyo;Rha, Sun-Young;Baek, Seung-Jae;Hwang, Jun-Yeon;Kim, Se-Heon
    • Korean Journal of Head & Neck Oncology
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    • v.27 no.2
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    • pp.190-197
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    • 2011
  • Background and Objective : Radiation resistance(RR) is one of main determinants of treatment outcome in oral squamous cell carcinoma(OSCC), but accurate prediction of RR is difficult. We aim to establish RR OSCC cell lines and identify genes related with RR by a measurement of altered gene expression after inducing RR. Material and Methods : OSCC cell lines, SCC15, SCC25 and QLL1, were treated with 2Gy radiation per session, and parts of them were alive in finally accumulated dosage of 60Gy through 30 times repetition of radiotherapy for inducing RR cell lines. We compared results of cDNA array and proteomics in non-radiated cell lines and RR cell lines to detect changes of gene expression. Western blot was used for the validation of results. Results : cDNA array revealed 265 commonly up-regulated genes and 268 commonly down-regulated genes in 3 RR cell lines comparing their non-radiated counterpart. Among them, 30 cancer related genes were obtained. Proteomics showed 51 commonly altered protein expressions in 3 RR cell lines and 18 cancer related proteins were obtained. Among the detected genes, we found NM23-H1 and PA2G4 were over-expressed in both cDNA array and proteomics. Western blot showed increased expression of NME1 in RR cell lines but not in PA2G4. Conclusion: We concluded that NM23-H1 may be a candidate of RR related gene and over-expression of NM23-H1 could be a biomarker to predict RR in OSCC.

A Standard [UC;AG] Vertical Block Code of Genetic Information 64 Trigram Codon (유전정보 64 Trigram Codon의 표준 [UC;AG] 수직 블록 Code)

  • Park, Ju-Yong;Lee, Sung-Kook;Lee, Moon-Ho
    • The Journal of the Institute of Internet, Broadcasting and Communication
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    • v.16 no.6
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    • pp.135-140
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    • 2016
  • In this paper, we analyze the [UC;AG] code which is genetic information standard DNA code, with 64 trigram. DNA which contains human genetic information, is a shape of adding three billion pairs of four bases which are A(adenine), C(cytosine), G(guanine) and T(thymine) to phosphoric acid and glucose. We present standard DNA code to 64 trigram which is $64{\times}4$ matrix with Kronecker product. This $64{\times}4$ matrix has double helix duplex property, and we can get the $4{\times}4$ matrix RNA code by removing the duplex of it. We present the DNA double helix to matrices and analysis the trigram array code of genetic information and the examples of it are presented in example 5, 6.

Identification of Genomic Aberrations by Array Comparative Genomic Hybridization in Patients with Aortic Dissections

  • Suh, Jong-Hui;Yoon, Jeong-Seob;Kwon, Jong-Bum;Kim, Hwan-Wook;Wang, Young-Pil
    • Journal of Chest Surgery
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    • v.44 no.2
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    • pp.123-130
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    • 2011
  • Background: The aim of the present study was to identify chromosomal loci that contribute to the pathogenesis of aortic dissection (AD) in a Korean population using array comparative genomic hybridization (CGH) and to confirm the results using real-time polymerase chain reaction (PCR). Materials and Methods: Eighteen patients with ADs were enrolled in this study. Genomic DNA was extracted from individual blood samples, and array CGH analyses were performed. Four corresponding genes with obvious genomic changes were analyzed using real-time PCR in order to assess the level of genomic imbalance identified by array CGH. Results: Genomic gains were most frequently detected at 8q24.3 (56%), followed by regions 7q35, 11q12.2, and 15q25.2 (50%). Genomic losses were most frequently observed at 4q35.2 (56%). Real-time PCR confirmed the results of the array CGH studies of the COL6A2, DGCR14, PCSK6, and SDHA genes. Conclusion: This is the first study to identify candidate regions by array CGH in patients with ADs. The identification of genes that may predispose an individual to AD may lead to a better understanding of the mechanism of AD formation. Further multicenter studies comparing cohorts of patients of different ethnicities are warranted.