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Evaluation of the Sequence Variability within the PCR Primer/Probe Target Regions of the SARS-CoV-2 Genome

Authors: Kashif Aziz Khan and Peter Cheung, date: 12/20/2020, view: 1605, Q&A: 0

[Abstract] Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2; initially named 2019-nCoV) is responsible for the recent coronavirus disease (COVID-19) pandemic, and polymerase chain reaction (PCR) is the current standard method for diagnosis from patient samples. As PCR assays are prone to sequence mismatches due to mutations in the viral genome, it ...

Measurement of the Length of the Integrated Donor DNA during Bacillus subtilis Natural Chromosomal Transformation

Authors: Ester Serrano and Begoña Carrasco, date: 08/20/2019, view: 1888, Q&A: 0

[Abstract] For natural transformation to occur, bacterial cells must first develop a programmed physiological state called competence. Competence in Bacillus subtilis, which occurs only in a fraction of cells, is a transient stress response that allows cells to take up DNA from the environment. During natural chromosomal transformation, the ...

Solid Phase PCR on 3D Microstructure ArrayChip for Pathogen Detection Application

Authors: Krishna Kant and Tien Anh Ngo, date: 08/05/2019, view: 3110, Q&A: 0

[Abstract] Advanced free angle photolithography (FAPL) is presented for making 3D supercritical angle fluorescence (SAF) microstructures and transfer them on to polymeric chips using injection molding technique for low-cost microfluidic devices embedded with optical sensing structures. A solid phase polymerase chain reaction (SP-PCR) is used as model ...

Quantification of Hepatitis B Virus Covalently Closed Circular DNA in Infected Cell Culture Models by Quantitative PCR

Authors: Bingqian Qu and Stephan Urban, date: 04/05/2019, view: 4275, Q&A: 0

[Abstract] Persistence of the human hepatitis B virus (HBV) requires the maintenance of covalently closed circular (ccc)DNA, the episomal genome reservoir in nuclei of infected hepatocytes. cccDNA elimination is a major aim in future curative therapies currently under development. In cell culture based in vitro studies, both hybridization- and ...

Rolling Circle Amplification to Screen Yam Germplasm for Badnavirus Infections and to Amplify and Characterise Novel Badnavirus Genomes

Authors: Moritz Bömer, Aliyu A. Turaki, Ajith I. Rathnayake, Gonçalo Silva, P. Lava Kumar and Susan E. Seal, date: 01/05/2018, view: 6608, Q&A: 0

[Abstract] Since the first discovery of badnaviruses (family Caulimoviridae, genus Badnavirus) in yam (Dioscorea spp.) germplasm in the 1970s (Harrison and Roberts, 1973), several hundred partial badnavirus reverse transcriptase (RT)-ribonuclease H (RNaseH) sequences have been characterised (Kenyon et al., 2008; Bousalem ...

Quantification of Densities of Bacterial Endosymbionts of Insects by Real-time PCR

Author: Daisuke Kageyama, date: 10/05/2017, view: 7599, Q&A: 0

[Abstract] Increased attention has been paid to the endosymbiotic bacteria of insects. Because most insect endosymbionts are uncultivable, quantitative PCR (qPCR) is a practical and convenient method to quantify endosymbiont titers. Here we report a protocol for real-time qPCR based on SYBR Green I fluorescence as well as some tips to prevent possible ...

Tagged Highly Degenerate Primer (THDP)-PCR for Community Analysis of Methane- and Ammonia-oxidizing Bacteria Based on Copper-containing Membrane-bound Monooxygenases (CuMMO)

Authors: Jian-Gong Wang, Fei Xia, Jemaneh Zeleke, Bin Zou and Zhe-Xue Quan, date: 06/20/2017, view: 6202, Q&A: 0

[Abstract] We describe a two-step PCR strategy using tagged highly degenerate primer (THDP-PCR) targeting copper-containing membrane-bound monooxygenases (CuMMO) genes for community analysis of methane- or ammonia-oxidizing bacteria. This strategy consists of a primary CuMMO gene-specific PCR followed by a secondary PCR with a tag as a single primer. This ...

Assaying the Effects of Splice Site Variants by Exon Trapping in a Mammalian Cell Line

Authors: Stuart W. Tompson and Terri L. Young, date: 05/20/2017, view: 9029, Q&A: 1

[Abstract] There are several in silico programs that endeavor to predict the functional impact of an individual’s sequence variation at splice donor/acceptor sites, but experimental confirmation is problematic without a source of RNA from the individual that carries the variant. With the aid of an exon trapping vector, such as pSPL3, an investigator ...

Identification of Natural Hybrids by SSR Markers in Mussaenda

Authors: Zhonglai Luo, Tingting Duan, Shuai Yuan, Shi Chen, Xiufeng Bai and Dianxiang Zhang, date: 07/05/2016, view: 7298, Q&A: 0

[Abstract] Detection of natural hybrids is of great significance for plant taxonomy, reproductive biology, and population genetic studies. Compared with methods depending on morphological characters, molecular markers provide reliable and much more accurate results. This protocol describes approaches employing microsatellite (SSR) markers to identify ...

3’ Rapid Amplification of cDNA Ends (3’ RACE) Using Arabidopsis Samples

Authors: Encarnación Rodríguez-Cazorla, Alfonso Andújar, Juan José Ripoll, Lindsay J. Bailey, Antonio Martínez-Laborda, Martin F. Yanofsky and Antonio Vera, date: 10/05/2015, view: 17781, Q&A: 0

[Abstract] Production of functional eukaryotic RNA is a very elaborate process that involves a complex interplay between transcription and various RNA processing activities, including splicing, 5’ capping, and 3’ cleavage and polyadenylation (Bentley, 2014). Accurate mapping of RNA ends provides a valuable tool to assess transcriptional and ...

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