scholarly journals Designing a Restriction Enzyme-free Method to Construct a MicroRNA Precursor gene for microRNA Cloning

2021 ◽  
Author(s):  
Mojdeh Amandadi ◽  
Mohammad Hashemabadi ◽  
hosseinali sasan
Keyword(s):  
High Performance ◽  
Expression Vector ◽  
Recombinant Expression ◽  
Restriction Enzymes ◽  
Enzymatic Digestion ◽  
Hek293 Cells ◽  
Microrna Gene ◽  
Cloning Method ◽  
Low Efficiency ◽  
Primer Sets

Abstract Common cloning strategies depend on the enzymatic digestion of the insert. In addition, the enzymatic digestion of PCR product ends by restriction enzymes is of low efficiency. These limitations are related to the need for enzymatic digestion to produce sticky ends in the insert sequence. Hence, in the present study, we aimed to present a new generation of pre-microRNA cloning method without using restriction enzymes for constructing pre-microRNA gene. In this strategy, by engineering an expression vector's sequence and designing two intelligent primer sets for two consecutive PCR reactions, the pre-microRNA sequence with appropriate restriction sites related to the expression vector was produced, without restriction enzymes. The recombinant expression vector was transfected into HEK293 cells, and microRNA-21 expression was assayed in these cells by real-time PCR, confirming the high efficacy of the presented cloning method. The present method can be an inexpensive and reliable method for microRNA precursor cloning by providing a high-performance protocol.

scholarly journals A novel target enrichment strategy in next-generation sequencing through 7-deaza-dGTP-resistant enzymatic digestion

2020 ◽  
Vol 13 (1) ◽  
Author(s):  
Peng Peng ◽  
Yanjuan Xu ◽  
Adrian M. Di Bisceglie ◽  
Xiaofeng Fan
Keyword(s):  
Next Generation Sequencing ◽  
Restriction Enzymes ◽  
Enzymatic Digestion ◽  
Target Enrichment ◽  
Next Generation ◽  
Two Phase ◽  
Low Efficiency ◽  
Viral Sequencing ◽  
Enrichment Strategy ◽  
Generation Sequencing

Abstract Objective Owing to the overwhelming dominance of human and commensal microbe sequences, low efficiency is a major concern in clinical viral sequencing using next-generation sequencing. DNA composed of 7-deaza-2′-deoxyguanosine 5′-triphosphate (c7dGTP), an analog of deoxyguanosine triphosphate (dGTP), is resistant to selective restriction enzymes. This characteristic has been utilized to develop a novel strategy for target enrichment in next-generation sequencing. Results The new enrichment strategy is named target enrichment via enzymatic digestion in next-generation sequencing (TEEDseq). It combined 7-deaza-2′-deoxyguanosine 5′-triphosphate (c7dGTP)-involved primer extension, splinter-assisted intracellular cyclization, c7dGTP)-resistant enzymatic digestion, and two-phase rolling cycle amplification. We first estimated c7dGTP for its efficiency in PCR amplification and its resistance to three restriction enzymes, AluI, HaeIII, and HpyCH4V. We then evaluated TEEDseq using a serum sample spiked with a 1311-bp hepatitis B virus (HBV) fragment. TEEDseq achieved an HBV on-target rate of 3.31 ± 0.39%, which was equivalent to 454× the enrichment of direct Illumina sequencing. Therefore, the current study has provided a concept proof for TEEDseq as an alternative option for clinical viral sequencing that requires an enrichment in next-generation sequencing.

Tetraphenylbenzene-based AIEgens: the Horizontally Oriented Emitters for highly Efficient Non-doped Deep Blue OLEDs and Host for High-Performance Hybrid WOLEDs

2020 ◽  
Author(s):  
Pengbo Han ◽  
Zeng Xu ◽  
Chengwei Lin ◽  
Dongge Ma ◽  
Anjun Qin ◽  
...  
Keyword(s):  
High Performance ◽  
General Strategy ◽  
Flat Panel Displays ◽  
Light Emitting ◽  
Horizontal Orientation ◽  
Deep Blue ◽  
Full Color ◽  
High Emission ◽  
White Oleds ◽  
Low Efficiency

Deep blue organic-emitting fluorophores are crucial for application in white lighting and full color flat-panel displays but emitters with high color quality and efficiency are rare. Herein, novel deep blue AIE luminogens (AIEgens) with various donor units and an acceptor of cyano substituted tetraphenylbenzene (TPB) cores were developed and used to fabricate non-doped deep blue and hybrid white organic light-emitting diodes (OLEDs). Benefiting from its high emission efficiency and high proportion of horizontally oriented dipoles in the film state, the non-doped deep blue device based on CN-TPB-TPA realized a maximum external quantum efficiency 7.27%, with a low efficiency roll-off and CIE coordinates of (0.15, 0.08). Moreover, efficient two-color hybrid warm white OLEDs (CIE<sub>x,y</sub> = 0.43, 0.45) were achieved using CN-TPB-TPA as the blue-emitting layer and phosphor doped host, which realized maximum current, power, external quantum efficiencies 58.0 cd A<sup>-1</sup>, 60.7 lm W<sup>-1</sup> and 19.1%, respectively. This work provides a general strategy to achieve high performance, stable deep blue and hybrid white OLEDs by construction of AIEgens with excellent horizontal orientation

Administration of the D-A structure and steric hindrance effect to construct efficient red emitters for high-performance OLEDs with low efficiency roll-off

2021 ◽  
Vol 192 ◽  
pp. 109398
Author(s):  
Guan-Yu Ding ◽  
Chun-Xiu Zang ◽  
Han Zhang ◽  
Zhong-Min Su ◽  
Guang-Fu Li ◽  
...  
Keyword(s):  
Steric Hindrance ◽  
High Performance ◽  
Low Efficiency

Synthesis of red/black phosphorus-based composite nanosheets with Z-scheme heterostructure for high-performance cancer phototherapy

Nanoscale ◽  
2021 ◽  
Author(s):  
Yong Kang ◽  
Zhengjun Li ◽  
Fengying Lu ◽  
Zhiguo Su ◽  
Xiaoyuan Ji ◽  
...  
Keyword(s):  
High Performance ◽  
Research Field ◽  
Black Phosphorus ◽  
Two Dimensional ◽  
The Poor ◽  
Low Efficiency ◽  
Poor Stability

Two dimensional black phosphorus nanosheets (BP NS) have attracted plenty of attentions in the research field of cancer photonic therapy. However, the poor stability and relatively low efficiency in reactive...

scholarly journals Characterization of LL25X, a Newly Isolated Anti-SIV Monoclonal Antibody, to Determine Binding Specificity

Elements ◽  
2017 ◽  
Vol 13 (1) ◽  
Author(s):  
Zackary Tajin Park
Keyword(s):  
Phage Display ◽  
Mammalian Cells ◽  
Expression Vector ◽  
Restriction Enzymes ◽  
Phage Display Library ◽  
Single Chain ◽  
Phage Display Technology ◽  
Mammalian Expression ◽  
Mammalian Expression Vector ◽  
Single Chain Fv

A phage display library was previously constructed from an SIV-infected rhesus macaque. Several single chain Fv (scFv), including SU24, SU343 and LL25X, were selected using phage display technology. Sequences corresponding to SU24, SU343 and LL25X were optimized for expression in a mammalian system and commercially synthesized. SU24 and SU343 had previously been cloned into a mammalian expression vector. In this study, we aimed to characterize the specificity of SU24, SU343, and LL25X.. The codon-optimized version of the scFv LL25X gene sequence was cloned into a mammalian expression vector (pCEP4).  LL25X DNA was amplified by PCR, and the PCR product and mammalian expression vector were both digested with KpnI/SapI restriction enzymes. Digested fragments were purified, and the fragments were ligated using T4DNA ligase. E. coli cells were transformed with the ligation reaction. Single colonies were selected on LB agar plates containing the selective antibiotic (ampicillin). Positive colonies were identified after DNA mini-preparation and test-digestion with KpnI and SapI. Sanger sequencing confirmed cloning results and DNA sequence accuracy. Following transfection of mammalian cells (293T), LL25X-Fc cells, and purifying our protein, the binding of LL25X-Fc to the SIV gp140 envelope protein was confirmed via ELISA and Western Blotting.

Cohosts with efficient host-to-emitter energy transfer for stable blue phosphorescent organic light-emitting diodes

2021 ◽  
Author(s):  
Soo-Ghang Ihn ◽  
Eun Suk Kwon ◽  
Yongsik Jung ◽  
Jong Soo Kim ◽  
Sungho Nam ◽  
...  
Keyword(s):  
Energy Transfer ◽  
High Performance ◽  
Light Emitting Diode ◽  
Organic Light Emitting Diodes ◽  
Operating Voltage ◽  
Light Emitting ◽  
Organic Light Emitting Diode ◽  
Organic Light ◽  
Low Efficiency ◽  
Blue Phosphorescent

We present a high-performance blue phosphorescent organic light-emitting diode exhibiting a low operating voltage (4.1 V), high external quantum efficiency (23.4%, at 500 cd m-2) with a low efficiency roll-off...

Rapid Integrated Method for Total Dietary Fiber

2021 ◽  
Keyword(s):  
Dietary Fiber ◽  
Resistant Starch ◽  
High Performance ◽  
Enzymatic Digestion ◽  
Soluble Dietary Fiber ◽  
Total Dietary Fiber ◽  
Food Ingredients ◽  
Insoluble Dietary Fiber ◽  
Integrated Method ◽  
Intestinal Digestion

This method determines total dietary fiber (TDF) in foods and food ingredients, as defined by Codex Alimentarius. The method measures soluble and insoluble dietary fiber, including resistant starch, as well as nondigestible oligosaccharides. In this method, enzymatic digestion is used to simulate human intestinal digestion. Insoluble dietary fiber (IDF) and soluble dietary fiber that precipitates in 78% ethanol (SDFP) are separated by filtration and quantified gravimetrically. Additionally, highly soluble oligosaccharides (SDFS) are quantified by chromatographic separation. TDF is reported as the sum of the gravimetric and high-performance liquid chromatography (HPLC) results. The digestion and chromatographic conditions of this method have been modified from those of AACC Approved Methods 32-45.01 and 32-50.01 in an attempt to better simulate human digestion and to allow for more exact quantitation.

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