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https://www.readbyqxmd.com/read/29789594/in-vitro-dna-scramble
#1
Yi Wu, Rui-Ying Zhu, Leslie A Mitchell, Lu Ma, Rui Liu, Meng Zhao, Bin Jia, Hui Xu, Yun-Xiang Li, Zu-Ming Yang, Yuan Ma, Xia Li, Hong Liu, Duo Liu, Wen-Hai Xiao, Xiao Zhou, Bing-Zhi Li, Ying-Jin Yuan, Jef D Boeke
The power of synthetic biology has enabled the expression of heterologous pathways in cells, as well as genome-scale synthesis projects. The complexity of biological networks makes rational de novo design a grand challenge. Introducing features that confer genetic flexibility is a powerful strategy for downstream engineering. Here we develop an in vitro method of DNA library construction based on structural variation to accomplish this goal. The "in vitro SCRaMbLE system" uses Cre recombinase mixed in a test tube with purified DNA encoding multiple loxPsym sites...
May 22, 2018: Nature Communications
https://www.readbyqxmd.com/read/29786943/integrating-a-comprehensive-dna-barcode-reference-library-with-a-global-map-of-yews-taxus-l-for-forensic-identification
#2
Jie Liu, Richard I Milne, Michael Möller, Guang-Fu Zhu, Lin-Jiang Ye, Ya-Huang Luo, Jun-Bo Yang, Moses Cheloti Wambulwa, Chun-Neng Wang, De-Zhu Li, Lian-Ming Gao
Rapid and accurate identification of endangered species is a critical component of bio-surveillance and conservation management, and potentially policing illegal trades. However, this is often not possible using traditional taxonomy, especially where only small or pre-processed parts of plants are available. Reliable identification can be achieved via a comprehensive DNA barcode reference library, accompanied by precise distribution data. However, these require extensive sampling at spatial and taxonomic scales, which has rarely been achieved for cosmopolitan taxa...
May 22, 2018: Molecular Ecology Resources
https://www.readbyqxmd.com/read/29786687/genome-wide-analysis-of-circular-rnas-in-bovine-cumulus-cells-treated-with-bmp15-and-gdf9
#3
Yao Fu, Hao Jiang, Jian-Bo Liu, Xu-Lei Sun, Zhe Zhang, Sheng Li, Yan Gao, Bao Yuan, Jia-Bao Zhang
Circular RNAs (circRNAs) are important members of the non-coding RNA family, and those relating to animal physiologies have been widely studied in recent years. This study aimed to explore the roles of circRNAs in the regulation of follicular development. We constructed four bovine cumulus cell cDNA libraries, including a negative control group (NC) and groups treated with BMP15, GDF9 and BMP15 + GDF9, and we sequenced the libraries on the Illumina HiSeq Xten platform. We identified 1706 circRNAs and screened for differential circRNA expression...
May 21, 2018: Scientific Reports
https://www.readbyqxmd.com/read/29786095/high-throughput-creation-and-functional-profiling-of-dna-sequence-variant-libraries-using-crispr-cas9-in-yeast
#4
Xiaoge Guo, Alejandro Chavez, Angela Tung, Yingleong Chan, Christian Kaas, Yi Yin, Ryan Cecchi, Santiago Lopez Garnier, Eric D Kelsic, Max Schubert, James E DiCarlo, James J Collins, George M Church
Construction and characterization of large genetic variant libraries is essential for understanding genome function, but remains challenging. Here, we introduce a Cas9-based approach for generating pools of mutants with defined genetic alterations (deletions, substitutions, and insertions) with an efficiency of 80-100% in yeast, along with methods for tracking their fitness en masse. We demonstrate the utility of our approach by characterizing the DNA helicase SGS1 with small tiling deletion mutants that span the length of the protein and a series of point mutations against highly conserved residues in the protein...
May 21, 2018: Nature Biotechnology
https://www.readbyqxmd.com/read/29785500/boosting-the-efficiency-of-site-saturation-mutagenesis-for-a-difficult-to-randomize-gene-by-a-two-step-pcr-strategy
#5
Aitao Li, Carlos G Acevedo-Rocha, Manfred T Reetz
Site-saturation mutagenesis (SSM) has been used in directed evolution of proteins for a long time. As a special form of saturation mutagenesis, it involves individual randomization at a given residue with formation of all 19 amino acids. To date, the most efficient embodiment of SSM is a one-step PCR-based approach using NNK codon degeneracy. However, in the case of difficult-to-randomize genes, SSM may not deliver all of the expected 19 mutants, which compels the user to invest further efforts by applying site-directed mutagenesis for the construction of the missing mutants...
May 21, 2018: Applied Microbiology and Biotechnology
https://www.readbyqxmd.com/read/29782009/construction-of-synthetic-phage-displayed-fab-library-with-tailored-diversity
#6
Ganggang Huang, Zhenwei Zhong, Shane Miersch, Sachdev S Sidhu, Shin-Chen Hou, Donghui Wu
Demand for monoclonal antibodies (mAbs) in basic research and medicine is increasing yearly. Hybridoma technology has been the dominant method for mAb development since its first report in 1975. As an alternative technology, phage display methods for mAb development are increasingly attractive since Humira, the first phage-derived antibody and one of the best-selling mAbs, was approved for clinical treatment of rheumatoid arthritis in 2002. As a non-animal based mAb development technology, phage display bypasses antigen immunogenicity, humanization, and animal maintenance that are required from traditional hybridoma technology based antibody development...
May 1, 2018: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/29781609/enhancement-and-analysis-of-human-anti-aflatoxin-b1-afb1-scfv-antibody-ligand-interaction-using-chain-shuffling
#7
Kuntalee Rangnoi, Kiattawee Choowongkomon, Richard O'Kennedy, Florian Rüker, Montarop Yamabhai
A human anti-aflatoxin B1 (AFB1) scFv antibody (yAFB1-c3), selected from a naïve human phage-displayed scFv library, was used as a template for improving and analysis of antibody-ligand interactions using the chain-shuffling technique. The variable heavy and variable light (VH/VL) shuffled library was constructed from the VH of 25 pre-selected clones recombined with the VL of yAFB1-c3 and vice versa. Affinity selection from these library demonstrated that the VH domain played an important role in the binding of scFv to free AFB1...
May 21, 2018: Journal of Agricultural and Food Chemistry
https://www.readbyqxmd.com/read/29775561/isolation-and-characterization-of-a-novel-anti-salbutamol-chicken-scfv-for-human-doping-urinalysis
#8
Warren Lee, Syed A Ali, Leow Chiuan Yee, Tan Soo Choon, Leow Chiuan Herng
Anti-salbutamol antibodies remain as important tools for the detection of salbutamol abuse in athletic doping. This study evaluated the feasibility and efficiency of the chicken (Gallus gallus domesticus) as an immunization host to generate anti-salbutamol scFv antibodies by phage display. A phage display antibody library was constructed from a single chicken immunized against salbutamol-KLH conjugate. After a stringent biopanning strategy, a novel scFv clone which was inhibited by free salbutamol recorded the highest affinity...
May 15, 2018: Analytical Biochemistry
https://www.readbyqxmd.com/read/29772229/polybacterial-community-analysis-in-human-conjunctiva-through-16s-rrna-gene-libraries
#9
Deepthi KrishnanNair Geetha, Jayasudha Rajagopalaboopathi, Girish Rameshan Nair, Manikandan Palanisamy, Ram Rammohan, Narendran Venkatapathy, Prabagaran Solai Ramatchandirane
The conjunctival sac of healthy human harbours a variety of microorganisms. When the eye is compromised, an occasional inadvertent spread happens to the adjacent tissue, resulting in bacterial ocular infections. Microbiological investigation of the conjunctival swab is one of the broadly used modality to study the aetiological agent of conjunctiva. However, most of the time such methods yield unsatisfactory results. Hence, the present study intends to identify the bacterial community in human conjunctiva of pre-operative subjects through 16S rRNA gene libraries...
May 14, 2018: Experimental Eye Research
https://www.readbyqxmd.com/read/29771937/construction-and-characterization-of-a-bacterial-artificial-chromosome-library-for-gossypium-mustelinum
#10
Yuling Liu, Baohong Zhang, Xinpeng Wen, Shulin Zhang, Yangyang Wei, Quanwei Lu, Zhen Liu, Kunbo Wang, Fang Liu, Renhai Peng
A bacterial artificial chromosome (BAC) library for G. mustelinum Miers ex G. Watt (AD4) was constructed. Intact nuclei from G. mustelinum (AD4) were used to isolate high molecular weight DNA, which was partially cleaved with Hind III and cloned into pSMART BAC (Hind III) vectors. The BAC library consisted of 208,182 clones arrayed in 542 384-microtiter plates, with an average insert size of 121.72 kb ranging from 100 to 150 kb. About 2% of the clones did not contain inserts. Based on an estimated genome size of 2372 Mb for G...
2018: PloS One
https://www.readbyqxmd.com/read/29767362/microrna-expression-analysis-next-generation-sequencing
#11
Poching Liu
MicroRNAs are a class of small noncoding RNAs that function as regulators involving in many biological processes. The evaluation of miRNAs and their targets has been aided by miRNA expression profiling studies including multiplex PCR, microarrays, and recent next-generation sequencing tools. Next-generation sequencing has enabled us to profile thousands of genes in a single experiment and overcome the background signal and cross-hybridization issues of microarrays. Next-generation sequencing also allows for the simultaneous confirmation of known miRNAs and discovery of new miRNAs, and significantly reduces costs while providing billions of nucleotide information within a single experiment...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29766833/human-micrornas-expression-profiles-in-influenza-b-virus-infected-cells-based-on-illumina-miseq-platform
#12
Kritsada Khongnomnan, Witthaya Poomipak, Kesmanee Praianantathavorn, Suthat Sangchuwong, Trairak Pisitkun, Yong Poovorawan, Sunchai Payungporn
BACKGROUND: Influenza B virus causes influenza-like illness in humans. MicroRNAs (miRNAs) are small non-coding RNAs regulating gene expression through mRNA degradation or translational repression. MiRNAs have evolved to regulate many cellular processes including the viral infection response. OBJECTIVE: This study aims to investigate the miRNA profiles of human cells infected with influenza B virus. METHODS: A549 cells were infected with influenza B viruses (MOI = 0...
May 14, 2018: MicroRNA
https://www.readbyqxmd.com/read/29766658/ampli-a-construction-set-for-paperfluidic-systems
#13
Elizabeth A Phillips, Anna K Young, Nikolas Albarran, Jonah Butler, Kaira Lujan, Kimberly Hamad-Schifferli, Jose Gomez-Marquez
The design and fabrication of reconfigurable, modular paperfluidics driven by a prefabricated reusable block library, asynchronous modular paperfluidic linear instrument-free (Ampli) block, are reported. The blocks are inspired by the plug-and-play modularity of electronic breadboards that lower prototyping barriers in circuit design. The resulting biochemical breadboard is a paperfluidic construction set that can be functionalized with chemical, biological, and electrical elements. Ampli blocks can form standard paperfluidic devices without any external instrumentation...
May 15, 2018: Advanced Healthcare Materials
https://www.readbyqxmd.com/read/29764756/straightforward-hit-identification-approach-in-fragment-based-discovery-of-bromodomain-containing-protein-4-brd4-inhibitors
#14
Petro Borysko, Yurii S Moroz, Oleksandr V Vasylchenko, Vasyl V Hurmach, Anastasia Starodubtseva, Natalia Stefanishena, Kateryna Nesteruk, Sergey Zozulya, Ivan S Kondratov, Oleksandr O Grygorenko
A combination approach of a fragment screening and "SAR by catalog" was used for the discovery of bromodomain-containing protein 4 (BRD4) inhibitors. Initial screening of 3695-fragment library against bromodomain 1 of BRD4 using thermal shift assay (TSA), followed by initial hit validation, resulted in 73 fragment hits, which were used to construct a follow-up library selected from available screening collection. Additionally, analogs of inactive fragments, as well as a set of randomly selected compounds were also prepared (3 × 3200 compounds in total)...
May 9, 2018: Bioorganic & Medicinal Chemistry
https://www.readbyqxmd.com/read/29763733/in-depth-profiling-of-mirna-regulation-in-the-body-wall-of-sea-cucumber-apostichopus-japonicus-during-skin-ulceration-syndrome-progression
#15
Hongjuan Sun, Zunchun Zhou, Ying Dong, Aifu Yang, Yongjia Pan, Jingwei Jiang, Zhong Chen, Xiaoyan Guan, Bai Wang, Shan Gao, Bei Jiang
MicroRNAs (miRNAs) are small non-coding RNAs that mediate mRNA degradation or translation repression. Previous study showed that the expression of miRNAs was significantly changed in the body wall of sea cucumber Apostichopus japonicus after skin ulceration syndrome (SUS) infection, which is a dynamic process. However, the critical miRNAs from body wall that involved in different infection stages of SUS remain unknown. In this study, four cDNA libraries were constructed with the body wall from healthy and three SUS-infected stages of A...
May 12, 2018: Fish & Shellfish Immunology
https://www.readbyqxmd.com/read/29757108/cognitively-active-older-adults-comprehension-and-metacomprehension-of-negated-text
#16
Sara J Margolin
Background/Study context: Previous research has demonstrated that negated text is universally difficult to understand, and while readers are aware of the difficulty, they are not always able to direct this awareness to improve their comprehension of negation. The present research aimed to determine whether this holds true for older adults, even while maintaining good cognitive function through reading activity. METHODS: The study used an online paradigm, where young (age range 19-24) and older (age range 60-87) adults read passages, rated their comprehension, and answered questions about what they read...
May 14, 2018: Experimental Aging Research
https://www.readbyqxmd.com/read/29754248/star-a-simple-tal-effector-assembly-reaction-using-isothermal-assembly
#17
Sabine Gogolok, Ute Köber, Steven M Pollard
Transcription activator-like effectors (TALEs) contain programmable DNA-binding domains that can be fused to various effectors to manipulate genetic sequences or transcriptional state. However, the construction of plasmids encoding the modular DNA-binding domain remains challenging due to their repetitive nature. Here, we describe methods for a simple TALE assembly reaction (STAR) that uses a 68-part plasmid library to create TALEs binding to 17 bp target sequences. Manual production of many tens of TALEs can be achieved using a simple 8 h protocol, with full length sequence-verified plasmids available within a few days...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29754244/ecoflex-a-multifunctional-moclo-kit-for-e-coli-synthetic-biology
#18
Hung-En Lai, Simon Moore, Karen Polizzi, Paul Freemont
Development of advanced synthetic biology tools is always in demand since they act as a platform technology to enable rapid prototyping of biological constructs in a high-throughput manner. EcoFlex is a modular cloning (MoClo) kit for Escherichia coli and is based on the Golden Gate principles, whereby Type IIS restriction enzymes (BsaI, BsmBI, BpiI) are used to construct modular genetic elements (biological parts) in a bottom-up approach. Here, we describe a collection of plasmids that stores various biological parts including promoters, RBSs, terminators, ORFs, and destination vectors, each encoding compatible overhangs allowing hierarchical assembly into single transcription units or a full-length polycistronic operon or biosynthetic pathway...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29754241/automated-visualization-of-genetic-designs-using-dnaplotlib
#19
Vittorio Bartoli, Daniel O R Dixon, Thomas E Gorochowski
Visualization of complex genetic systems can help efficiently communicate important design features and clearly illustrate overall structures. To aid in the creation of such diagrams, standards such as the Synthetic Biology Open Language Visual (SBOLv) have been established to ensure that specific symbols and shapes convey the same meaning for genetic parts across the field. Here, we describe several ways that the computational tool DNAplotlib can be used to automate the generation of SBOLv standard-compliant diagrams covering simple genetic designs to large libraries of genetic constructs...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29754226/construction-of-crispr-libraries-for-functional-screening
#20
Carsten P Carstens, Katherine A Felts, Sarah E Johns
Identification of gene function has been aided by the ability to generate targeted gene knockouts or transcriptional repression using the CRISPR/CAS9 system. Using pooled libraries of guide RNA expression vectors that direct CAS9 to a specific genomic site allows identification of genes that are either enriched or depleted in response to a selection scheme, thus linking the affected gene to the chosen phenotype. The quality of the data generated by the screening is dependent on the quality of the guide RNA delivery library with regards to error rates and especially evenness of distribution of the guides...
2018: Methods in Molecular Biology
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