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Sequence library construction

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https://www.readbyqxmd.com/read/28448969/synthetic-switchable-enzymes
#1
Vic Norris, Sergey N Krylov, Pratul K Agarwal, Glenn J White
The construction of switchable, radiation-controlled, aptameric enzymes - "swenzymes" - is, in principle, feasible. We propose a strategy to make such catalysts from 2 (or more) aptamers each selected to bind specifically to one of the substrates in, for example, a 2-substrate reaction. Construction of a combinatorial library of candidate swenzymes entails selecting a set of a million aptamers that bind one substrate and a second set of a million aptamers that bind the second substrate; the aptamers in these sets are then linked pairwise by a linker, thus bringing together the substrates...
April 28, 2017: Journal of Molecular Microbiology and Biotechnology
https://www.readbyqxmd.com/read/28446605/crispr-cas9-mediated-gene-knockout-screens-and-target-identification-via-whole-genome-sequencing-uncover-host-genes-required-for-picornavirus-infection
#2
Heon Seok Kim, Kyungjin Lee, Sangsu Bae, Jeongbin Park, Chong-Kyo Lee, Meehyein Kim, Eunji Kim, Minju Kim, Seokjoong Kim, Chonsaeng Kim, Jin-Soo Kim
Several groups have used genome-wide libraries of lentiviruses encoding small-guide RNAs (sgRNAs) for genetic screens. In most cases, sgRNA expression cassettes are integrated into cells by using lentiviruses, and target genes are statistically estimated by the readout of sgRNA sequences after targeted sequencing. We present a new virus-free method for human gene-knockout screens using a genome-wide library of CRISPR/Cas9 sgRNAs based on plasmids, and target gene identification via whole-genome sequencing (WGS) confirming authentic mutations rather than statistical estimating through targeted amplicon sequencing...
April 26, 2017: Journal of Biological Chemistry
https://www.readbyqxmd.com/read/28445507/various-mutations-compensate-for-a-deleterious-lacz%C3%AE-insert-in-the-replication-enhancer-of-m13-bacteriophage
#3
Emily M Zygiel, Karen A Noren, Marta A Adamkiewicz, Richard J Aprile, Heather K Bowditch, Christine L Carroll, Maria Abigail S Cerezo, Adelle M Dagher, Courtney R Hebert, Lauren E Hebert, Gloria M Mahame, Stephanie C Milne, Kelly M Silvestri, Sara E Sutherland, Alexandria M Sylvia, Caitlyn N Taveira, David J VanValkenburgh, Christopher J Noren, Marilena Fitzsimons Hall
M13 and other members of the Ff class of filamentous bacteriophages have been extensively employed in myriad applications. The Ph.D. series of phage-displayed peptide libraries were constructed from the M13-based vector M13KE. As a direct descendent of M13mp19, M13KE contains the lacZα insert in the intergenic region between genes IV and II, where it interrupts the replication enhancer of the (+) strand origin. Phage carrying this 816-nucleotide insert are viable, but propagate in E. coli at a reduced rate compared to wild-type M13 phage, presumably due to a replication defect caused by the insert...
2017: PloS One
https://www.readbyqxmd.com/read/28444590/transcripts-of-pectin-degrading-enzymes-and-isolation-of-complete-cdna-sequence-of-a-pectate-lyase-gene-induced-by-coffee-white-stem-borer-xylotrechus-quadripes-in-the-bark-tissue-of-coffea-canephora-robusta-coffee
#4
Kosaraju Bharathi, P Santosh, H L Sreenath
Of the two commercially cultivated coffee (Coffea) species, C. arabica (arabica) is highly susceptible and C. canephora (robusta) is highly resistant to the insect pest Xylotrechus quadripes (Coleoptera: Cerambycidae), commonly known as coffee white stem borer (CWSB). We constructed a forward-subtracted cDNA library by Suppression Subtractive Hybridization (SSH) from robusta bark tissue for profiling genes induced by CWSB infestation. Among the 265 unigenes of the SSH EST library, 7 unigenes (5 contigs and 2 singletons) matching different pectin-degrading enzymes were discovered...
May 2017: 3 Biotech
https://www.readbyqxmd.com/read/28439859/genome-wide-profiling-of-histone-modifications-and-histone-variants-in-arabidopsis-thaliana-and-marchantia-polymorpha
#5
Ramesh Yelagandula, Akihisa Osakabe, Elin Axelsson, Frederic Berger, Tomokazu Kawashima
Histone modifications and histone variants barcode the genome and play major roles in epigenetic regulations. Chromatin immunoprecipitation (ChIP) coupled with next-generation sequencing (NGS) is a well-established method to investigate the landscape of epigenetic marks at a genomic level. Here, we describe procedures for conducting ChIP, subsequent NGS library construction, and data analysis on histone modifications and histone variants in Arabidopsis thaliana. We also describe an optimized nuclear isolation procedure to prepare chromatin for ChIP in the liverwort, Marchantia polymorpha, which is the emerging model plant ideal for evolutionary studies...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28439825/small-rna-profiling-by-next-generation-sequencing-using-high-definition-adapters
#6
Martina Billmeier, Ping Xu
Small RNAs (sRNAs) as key regulators of gene expression play fundamental roles in many biological processes. Next-generation sequencing (NGS) has become an important tool for sRNA discovery and profiling. However, NGS data often show bias for or against certain sequences which is mainly caused by adapter oligonucleotides that are ligated to sRNAs more or less efficiently by RNA ligases. In order to reduce ligation bias, High-definition (HD) adapters for the Illumina sequencing platform were developed. However, a large amount of direct 5' and 3' adapter ligation products are often produced when the current commercially available kits are used for cloning with HD adapters...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28439479/simple-sequence-repeat-markers-for-k%C3%A4-nuka-kunzea-spp-myrtaceae-present-in-new-zealand
#7
Dagmar F Goeke, Caroline M Mitchell, Claudia Lange, Gary J Houliston
PREMISE OF THE STUDY: We developed simple sequence repeat (SSR) markers to facilitate population genetic studies on kānuka (Kunzea spp.; Myrtaceae). METHODS AND RESULTS: A shotgun sequencing library was constructed from leaf material of K. robusta using a Roche 454 Junior sequencer, and a total of 3174 putative SSR regions were identified. Sixteen polymorphic markers were optimized for multiplex PCR on 10 endemic New Zealand Kunzea species. Each of these loci cross-amplified in all tested species...
April 2017: Applications in Plant Sciences
https://www.readbyqxmd.com/read/28437612/combinatorial-library-based-on-restriction-enzyme-mediated-modular-assembly
#8
Cuiping Ma, Chao Liang, Yifan Wang, Mei Pan, Qianqian Jiang, Chao Shi
Combinatorial approaches in directed evolution were proven to be more efficient for exploring sequence space and innovating the function of protein. We presented modular assembly of secondary structures (MASS) for constructing a combinatorial library. In this approach, secondary structure elements were extracted from natural existing proteins, the common linkers were flanking secondary structure elements, then digested by Hinf I restriction endonuclease that was used in the construction of combinatorial library for the first time...
April 24, 2017: ACS Combinatorial Science
https://www.readbyqxmd.com/read/28432352/low-cost-low-bias-and-low-input-rna-seq-with-high-experimental-verifiability-based-on-semiconductor-sequencing
#9
Zhibiao Mai, Chuanle Xiao, Jingjie Jin, Gong Zhang
Low-input RNA-seq is powerful to represent the gene expression profiles with limited number of cells, especially when single-cell variations are not the aim. However, pre-amplification-based and molecule index-based library construction methods boost bias or require higher throughput. Here we demonstrate a simple, low-cost, low-bias and low-input RNA-seq with ion torrent semiconductor sequencing (LIEA RNA-seq). We also developed highly accurate and error-tolerant spliced mapping algorithm FANSe2splice to accurately map the single-ended reads to the reference genome with better experimental verifiability than the previous spliced mappers...
April 21, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28432195/n-terminal-proteomics-assisted-profiling-of-the-unexplored-translation-initiation-landscape-in-arabidopsis-thaliana
#10
Patrick Willems, Elvis Ndah, Veronique Jonckheere, Simon Stael, Adriaan Sticker, Lennart Martens, Frank Van Breusegem, Kris Gevaert, Petra Van Damme
Proteogenomics is an emerging research field yet lacking a uniform method of analysis. Proteogenomic studies in which N-terminal proteomics and ribosome profiling are combined, suggest that a high number of protein start sites are currently missing in genome annotations. We constructed a proteogenomic pipeline specific for the analysis of N-terminal proteomics data, with the aim of discovering novel translational start sites outside annotated protein coding regions. In summary, unidentified MS/MS spectra were matched to a specific N-terminal peptide library encompassing protein N-termini encoded in the Arabidopsis thaliana genome...
April 21, 2017: Molecular & Cellular Proteomics: MCP
https://www.readbyqxmd.com/read/28431567/transcriptome-profiling-of-elymus-sibiricus-an-important-forage-grass-in-qinghai-tibet-plateau-reveals-novel-insights-into-candidate-genes-that-potentially-connected-to-seed-shattering
#11
Wengang Xie, Junchao Zhang, Xuhong Zhao, Zongyu Zhang, Yanrong Wang
BACKGROUND: Elymus sibiricus is an important forage grass in semi-arid regions, but it is difficult to grow for commercial seed production due to high seed shattering. To better understand the underlying mechanism and explore the putative genes related to seed shattering, we conducted a combination of morphological, histological, physiochemical and transcriptome analysis on two E. sibiricus genotypes (XH09 and ZhN03) that have contrasting seed shattering. RESULTS: The results show that seed shattering is generally caused by a degradation of the abscission layer...
April 21, 2017: BMC Plant Biology
https://www.readbyqxmd.com/read/28431015/large-scale-collection-of-full-length-cdna-and-transcriptome-analysis-in-hevea-brasiliensis
#12
Yuko Makita, Kiaw Kiaw Ng, G Veera Singham, Mika Kawashima, Hideki Hirakawa, Shusei Sato, Ahmad Sofiman Othman, Minami Matsui
Natural rubber has unique physical properties that cannot be replaced by products from other latex-producing plants or petrochemically produced synthetic rubbers. Rubber from Hevea brasiliensis is the main commercial source for this natural rubber that has a cis-polyisoprene configuration. For sustainable production of enough rubber to meet demand elucidation of the molecular mechanisms involved in the production of latex is vital. To this end, we firstly constructed rubber full-length cDNA libraries of RRIM 600 cultivar and sequenced around 20,000 clones by the Sanger method and over 15,000 contigs by Illumina sequencer...
April 1, 2017: DNA Research: An International Journal for Rapid Publication of Reports on Genes and Genomes
https://www.readbyqxmd.com/read/28429252/analysis-of-global-gene-expression-profiles-during-the-flowering-initiation-process-of-lilium%C3%A2-%C3%A3-%C3%A2-formolongi
#13
Yu-Fan Li, Ming-Fang Zhang, Meng Zhang, Gui-Xia Jia
The onset of flowering is critical for the reproductive development of plants. Lilium × formolongi is a lily hybrid that flowers within a year after sowing. We successfully identified four important stages during vegetative growth and flowering initiation of L. × formolongi under long day conditions. The plant tissues from the four stages were used in a genome-wide transcriptional analysis to investigate stage-specific changes of gene expression in L. × formolongi. In total, the sequence reads of the four RNA-sequencing libraries were assembled into 52,824 unigenes, of which 37,031 (70...
April 20, 2017: Plant Molecular Biology
https://www.readbyqxmd.com/read/28426424/cdna-library-construction-of-two-human-demodexspecies
#14
DongLing Niu, RuiLing Wang, YaE Zhao, Rui Yang, Li Hu, YuYang Lei, WeiChao Dan
The research of Demodex, a type of pathogen causing various dermatoses in animals and human beings, is lacking at RNA level. This study aims at extracting RNA and constructing cDNA library for Demodex. First, P. cuniculiand D. farinaewere mixed to establish homogenization method for RNA extraction. Second, D. folliculorumand D. breviswere collected and preserved in Trizol, which were mixed with D. farinaerespectively to extract RNA. Finally, cDNA library was constructed and its quality was assessed. The results indicated that for D...
June 1, 2017: Acta Parasitologica
https://www.readbyqxmd.com/read/28424372/effects-of-drought-stress-on-global-gene-expression-profile-in-leaf-and-root-samples-of-dongxiang-wild-rice-oryza-rufipogon
#15
Fantao Zhang, Yi Zhou, Meng Zhang, Xiangdong Luo, Jiankun Xie
Drought is a serious constraint to rice production throughout the world, and although Dongxiang wild rice ( Oryza rufipogon , DXWR) possesses a high degree of drought resistance, the underlying mechanisms of this trait remains unclear. In the present study, cDNA libraries were constructed from the leaf and root tissues of drought-stressed and untreated DXWR seedlings, and transcriptome sequencing was performed with the goal of elucidating the molecular mechanisms involved in drought stress response. The results indicated that 11,231 transcripts were differentially expressed in the leaves (4,040 up-regulated and 7,191 down-regulated) and 7,025 transcripts were differentially expressed in the roots (3,097 up-regulated and 3,928 down-regulated)...
April 19, 2017: Bioscience Reports
https://www.readbyqxmd.com/read/28423600/systematic-screening-of-isogenic-cancer-cells-identifies-dusp6-as-context-specific-synthetic-lethal-target-in-melanoma
#16
Stephanie Wittig-Blaich, Rainer Wittig, Steffen Schmidt, Stefan Lyer, Melanie Bewerunge-Hudler, Sabine Gronert-Sum, Olga Strobel-Freidekind, Carolin Müller, Markus List, Aleksandra Jaskot, Helle Christiansen, Mathias Hafner, Dirk Schadendorf, Ines Block, Jan Mollenhauer
Next-generation sequencing has dramatically increased genome-wide profiling options and conceptually initiates the possibility for personalized cancer therapy. State-of-the-art sequencing studies yield large candidate gene sets comprising dozens or hundreds of mutated genes. However, few technologies are available for the systematic downstream evaluation of these results to identify novel starting points of future cancer therapies.We improved and extended a site-specific recombination-based system for systematic analysis of the individual functions of a large number of candidate genes...
April 4, 2017: Oncotarget
https://www.readbyqxmd.com/read/28422724/utilizing-combinatorial-engineering-to-develop-tie2-targeting-antagonistic-angiopoetin-2-ligands-as-candidates-for-anti-angiogenesis-therapy
#17
Tomer Shlamkovich, Lidan Aharon, William A Barton, Niv Papo
In many human cancers, the receptor tyrosine kinase (RTK) Tie2 plays important roles in mediating proliferation, survival, migration and angiogenesis. Thus, molecules that could potently inhibit activation of the Tie2 receptor would have a significant impact on cancer therapy. Nevertheless, attempts to develop Tie2-targeted inhibitors have met with little success, and there is currently no FDA-approved therapeutic selectively targeting Tie2. We used a combinatorial protein engineering approach to develop a new generation of angiopoietin (Ang) 2-derived Tie2 antagonists as potential cancer therapeutics and as tools to study angiogenesis...
April 4, 2017: Oncotarget
https://www.readbyqxmd.com/read/28419064/the-application-of-next-generation-sequencing-for-mutation-detection-in-autosomal-dominant-hereditary-hearing-impairment
#18
Nicolas Gürtler, Benno Röthlisberger, Katja Ludin, Christoph Schlegel, Anil K Lalwani
OBJECTIVE: Identification of the causative mutation using next-generation sequencing in autosomal-dominant hereditary hearing impairment, as mutation analysis in hereditary hearing impairment by classic genetic methods, is hindered by the high heterogeneity of the disease. PATIENTS: Two Swiss families with autosomal-dominant hereditary hearing impairment. INTERVENTION: Amplified DNA libraries for next-generation sequencing were constructed from extracted genomic DNA, derived from peripheral blood, and enriched by a custom-made sequence capture library...
April 17, 2017: Otology & Neurotology
https://www.readbyqxmd.com/read/28415833/molecular-characterization-and-biodiversity-of-a-putative-chlorotoxin-from-the-iranian-yellow-scorpion-odontobuthus-doriae
#19
Maryam Naderi Soorki, Amir Jalali, Hamid Galehdari
BAckground: Chloride channels have already been over-expressed in the different types of cancer. Chlorotoxins, as the blocking agent of these channels, have been indicated to be an effective drug against tumors. In this study, we characterized a putative chlorotoxin from a cDNA library of the venom glands obtained from the Iranian scorpion Odontobuthus doriae. Methods: A cDNA library was constructed from venom gland transcriptome of six scorpions. The cDNA encoding Odontobuthus doriae chlorotoxin was isolated from the library, and its putative peptide was characterized by some bioinformatics software such as protein blast, SignalP4...
April 18, 2017: Iranian Biomedical Journal
https://www.readbyqxmd.com/read/28408977/mitogenomes-of-giant-skipper-butterflies-reveal-an-ancient-split-between-deep-and-shallow-root-feeders
#20
Jing Zhang, Qian Cong, Xiao-Ling Fan, Rongjiang Wang, Min Wang, Nick V Grishin
Background: Giant-Skipper butterflies from the genus Megathymus are North American endemics. These large and thick-bodied Skippers resemble moths and are unique in their life cycles. Grub-like at the later stages of development, caterpillars of these species feed and live inside yucca roots. Adults do not feed and are mostly local, not straying far from the patches of yucca plants. Methods: Pieces of muscle were dissected from the thorax of specimens and genomic DNA was extracted (also from the abdomen of a specimen collected nearly 60 years ago)...
2017: F1000Research
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