scholarly journals Family-Specific Degenerate Primer Design: A Tool to Design Consensus Degenerated Oligonucleotides

2013 ◽  
Vol 2013 ◽  
pp. 1-9 ◽  
Author(s):  
Javier Alonso Iserte ◽  
Betina Ines Stephan ◽  
Sandra Elizabeth Goñi ◽  
Cristina Silvia Borio ◽  
Pablo Daniel Ghiringhelli ◽  
...  
Keyword(s):  
Computer Software ◽  
Pcr Primers ◽  
Primer Design ◽  
Degenerate Primer ◽  
Related Protein ◽  
Degenerate Primers ◽  
Degenerate Pcr ◽  
New Family ◽  
Starting Point ◽  
Wide Range

Designing degenerate PCR primers for templates of unknown nucleotide sequence may be a very difficult task. In this paper, we present a new method to design degenerate primers, implemented in family-specific degenerate primer design (FAS-DPD) computer software, for which the starting point is a multiple alignment of related amino acids or nucleotide sequences. To assess their efficiency, four different genome collections were used, covering a wide range of genomic lengths: Arenavirus ( nucleotides), Baculovirus ( to  bp), Lactobacillus sp. ( to  bp), and Pseudomonas sp. ( to  bp). In each case, FAS-DPD designed primers were tested computationally to measure specificity. Designed primers for Arenavirus and Baculovirus were tested experimentally. The method presented here is useful for designing degenerate primers on collections of related protein sequences, allowing detection of new family members.

Classification and review of free PCR primer design software

2020 ◽  
Author(s):  
Jingwen Guo ◽  
David Starr ◽  
Huazhang Guo
Keyword(s):  
Computer Software ◽  
Pcr Primers ◽  
Primer Design ◽  
Degenerate Primers ◽  
Targeted Next Generation Sequencing ◽  
Human Element ◽  
Reverse Transcription Quantitative Pcr ◽  
New Gene ◽  
Design Software ◽  
Pcr Primer

Abstract Motivation Polymerase chain reaction (PCR) has been a revolutionary biomedical advancement. However, for PCR to be appropriately used, one must spend a significant amount of effort on PCR primer design. Carefully designed PCR primers not only increase sensitivity and specificity, but also decrease effort spent on experimental optimization. Computer software removes the human element by performing and automating the complex and rigorous calculations required in PCR primer design. Classification and review of the available software options and their capabilities should be a valuable resource for any PCR application. Results This article focuses on currently available free PCR primer design software and their major functions (https://pcrprimerdesign.github.io/). The software are classified according to their PCR applications, such as Sanger sequencing, reverse transcription quantitative PCR, single nucleotide polymorphism detection, splicing variant detection, methylation detection, microsatellite detection, multiplex PCR and targeted next generation sequencing, and conserved/degenerate primers to clone orthologous genes from related species, new gene family members in the same species, or to detect a group of related pathogens. Each software is summarized to provide a technical review of their capabilities and utilities. Contact [email protected]

scholarly journals Occurrence of a Sequence in Marine Cyanophages Similar to That of T4 g20 and Its Application to PCR-Based Detection and Quantification Techniques

1998 ◽  
Vol 64 (6) ◽  
pp. 2051-2060 ◽  
Author(s):  
Nicholas J. Fuller ◽  
William H. Wilson ◽  
Ian R. Joint ◽  
Nicholas H. Mann
Keyword(s):  
Sequence Analysis ◽  
Pcr Primers ◽  
Degenerate Primers ◽  
Degenerate Pcr ◽  
Gene Encoding ◽  
Significant Similarity ◽  
Pcr Products ◽  
Seawater Samples ◽  
Log Linear ◽  
Correct Size

ABSTRACT Viruses are ubiquitous components of marine ecosystems and are known to infect unicellular phycoerythrin-containing cyanobacteria belonging to the genus Synechococcus. A conserved region from the cyanophage genome was identified in three genetically distinct cyanomyoviruses, and a sequence analysis revealed that this region exhibited significant similarity to a gene encoding a capsid assembly protein (gp20) from the enteric coliphage T4. The results of a comparison of gene 20 sequences from three cyanomyoviruses and T4 allowed us to design two degenerate PCR primers, CPS1 and CPS2, which specifically amplified a 165-bp region from the majority of cyanomyoviruses tested. A competitive PCR (cPCR) analysis revealed that cyanomyovirus strains could be accurately enumerated, and it was demonstrated that quantification was log-linear over ca. 3 orders of magnitude. Different calibration curves were obtained for each of the three cyanomyovirus strains tested; consequently, cPCR performed with primers CPS1 and CPS2 could lead to substantial inaccuracies in estimates of phage abundance in natural assemblages. Further sequence analysis of cyanomyovirus gene 20 homologs would be necessary in order to design primers which do not exhibit phage-to-phage variability in priming efficiency. It was demonstrated that PCR products of the correct size could be amplified from seawater samples following 100× concentration and even directly without any prior concentration. Hence, the use of degenerate primers in PCR analyses of cyanophage populations should provide valuable data on the diversity of cyanophages in natural assemblages. Further optimization of procedures may ultimately lead to a sensitive assay which can be used to analyze natural cyanophage populations both quantitatively (by cPCR) and qualitatively following phylogenetic analysis of amplified products.

scholarly journals DegePrime, a Program for Degenerate Primer Design for Broad-Taxonomic-Range PCR in Microbial Ecology Studies

2014 ◽  
Vol 80 (16) ◽  
pp. 5116-5123 ◽  
Author(s):  
Luisa W. Hugerth ◽  
Hugo A. Wefer ◽  
Sverker Lundin ◽  
Hedvig E. Jakobsson ◽  
Mathilda Lindberg ◽  
...  
Keyword(s):  
Pcr Amplification ◽  
Pcr Primers ◽  
Primer Design ◽  
Taxonomic Composition ◽  
Degenerate Primer ◽  
Rrna Gene ◽  
Multiple Sequence ◽  
High Coverage ◽  
Shotgun Metagenomics ◽  
Taxonomic Range

ABSTRACTThe taxonomic composition of a microbial community can be deduced by analyzing its rRNA gene content by, e.g., high-throughput DNA sequencing or DNA chips. Such methods typically are based on PCR amplification of rRNA gene sequences using broad-taxonomic-range PCR primers. In these analyses, the use of optimal primers is crucial for achieving an unbiased representation of community composition. Here, we present the computer program DegePrime that, for each position of a multiple sequence alignment, finds a degenerate oligomer of as high coverage as possible and outputs its coverage among taxonomic divisions. We show that our novel heuristic, which we call weighted randomized combination, performs better than previously described algorithms for solving the maximum coverage degenerate primer design problem. We previously used DegePrime to design a broad-taxonomic-range primer pair that targets the bacterial V3-V4 region (341F-805R) (D. P. Herlemann, M. Labrenz, K. Jurgens, S. Bertilsson, J. J. Waniek, and A. F. Andersson, ISME J. 5:1571–1579, 2011,http://dx.doi.org/10.1038/ismej.2011.41), and here we use the program to significantly increase the coverage of a primer pair (515F-806R) widely used for Illumina-based surveys of bacterial and archaeal diversity. By comparison with shotgun metagenomics, we show that the primers give an accurate representation of microbial diversity in natural samples.

scholarly journals In silico design and validation of a highly degenerate primer pair: a systematic approach

2020 ◽  
Vol 18 (1) ◽  
Author(s):  
Prosper Obed Chukwuemeka ◽  
Haruna Isiyaku Umar ◽  
Oluwatoyin Folake Olukunle ◽  
Oluwaseyi Matthew Oretade ◽  
Christopher Busayo Olowosoke ◽  
...  
Keyword(s):  
In Silico ◽  
Biological Activities ◽  
Positive Control ◽  
Primer Design ◽  
Extensive Study ◽  
Degenerate Primer ◽  
Degenerate Primers ◽  
Software Program ◽  
Optimal Efficiency ◽  
Enzymatic Degradability

Abstract Background The techniques of amplifying genetic materials have enabled the extensive study of several biological activities outside the biological milieu of living systems. More recently, this approach has been extended to amplify population of genes, from evolutionarily related gene family for detection and evaluation of microbial consortial with several unique potentialities (e.g., enzymatic degradability). Conceivably, primer mixtures containing substitutions of different bases at specific sites (degenerate primers) have enabled the amplification of these genes in PCR reaction. However, the degenerate primer design problem (DPD) is a constraint to designing this kind of primer. To date, different algorithms now exist to solve various versions of DPD problem, many of which, only few addresses and satisfy the criteria to design primers that can extensively cover high through-put sequences while striking the balance between specificity and efficiency. The highly degenerate primer (HYDEN) design software program primarily addresses this variant of DPD problem termed “maximum coverage-degenerate primer design (MC-DPD)” and its heuristics have been substantiated for optimal efficiency from significant successes in PCR. In spite of the premium presented for designing degenerate primers, literature search has indicated relatively little use of its heuristics. This has been thought to result from the complexity of the program since it is run only by command-line, hence limiting its accessibility. To solve this problem, researchers have optionally considered the manual design of degenerate primers or design through software programs that provides accessibility through a graphical user interface (GUI). Realizing this, we have attempted in this study to provide a user-friendly approach for researchers with little or no background in bioinformatics to design degenerate primers using HYDEN Results Virtual Tests of our designed degenerate primer pair through in silico PCR substantiated the correspondence between efficiency and coverage with the target sequences as pre-defined by the initial HYDEN output, thereby validating the potentials of HYDEN to effectively solve the MC-DPD problem. Additionally, the designed primer-pair mechanistically amplified all sequences used as a positive control with no amplification observed in the negative controls. Conclusion In this study, we provided a turnkey protocol to simplify the design of degenerate primers using the heuristics of the HYDEN software program.

A new family of NxOy pyridine-containing macrocycles: Synthesis and characterization of their Y(III), Ln(III), Zn(II), and Cd(II) coordination compounds

2004 ◽  
Vol 82 (3) ◽  
pp. 437-447 ◽  
Author(s):  
Carlos Lodeiro ◽  
Rufina Bastida ◽  
Emilia Bértolo ◽  
Adolfo Rodríguez
Keyword(s):  
Schiff Base ◽  
Macrocyclic Ligand ◽  
Macrocyclic Ligands ◽  
New Family ◽  
Pendant Arm ◽  
Starting Point ◽  
Wide Range ◽  
Imine Bond ◽  
Pendant Arms

Reaction between 2,6-bis(2-formylphenoxymethyl)pyridine and N,N-bis(3-aminopropyl)methylamine or tris(2-aminoethyl)amine has been used as the starting point for the synthesis of seven oxa-aza macrocyclic ligands, five of them never reported previously. They all feature different pendant arms, which provide a wide range of coordination possibilities. The Schiff base macrocycles L1 and L4 and their reduced ligands L2 and L5 are derived from 2,6-bis(2-formylphenoxymethyl)pyridine and tris(2-aminoethyl)amine or N,N-bis(3-aminopropyl)methylamine, respectively. The reaction of L1 with salicylaldehyde forms L3, which features an imine bond in the pendant arm. The ligand L5 has been the precursor for the pendant-armed L6 and L7, by alkylation of the free NH groups with methyl-imidazole or methyl-indole. By a template or a nontemplate approach, we have synthesized different mono- and dinuclear complexes with Y(III), Ln(III), Zn(II), and Cd(II) cations. Both the free macrocyclic ligands and their corresponding metal complexes have been characterized by microanalysis, IR, UV–vis, 1H and 13C NMR spectroscopy, FAB mass spectrometry, MS electrospray, and conductivity measurements.Key words: Schiff-base macrocycle, template synthesis, macrocyclic ligand complexes, lanthanide(III) complexes.

Discovery of Antibacterial Agents Inhibiting the Energy-Coupling Factor (ECF) Transporters by Structure-Based Virtual Screening

2020 ◽  
Author(s):  
Eleonora Diamanti ◽  
Inda Setyawati ◽  
Spyridon Bousis ◽  
leticia mojas ◽  
lotteke Swier ◽  
...  
Keyword(s):  
Virtual Screening ◽  
Antibacterial Agents ◽  
Antimicrobial Agents ◽  
Energy Coupling ◽  
Coupling Factor ◽  
Design Synthesis ◽  
Screening Design ◽  
Starting Point ◽  
Wide Range ◽  
Vitamin Uptake

Here, we report on the virtual screening, design, synthesis and structure–activity relationships (SARs) of the first class of selective, antibacterial agents against the energy-coupling factor (ECF) transporters. The ECF transporters are a family of transmembrane proteins involved in the uptake of vitamins in a wide range of bacteria. Inhibition of the activity of these proteins could reduce the viability of pathogens that depend on vitamin uptake. Because of their central role in the metabolism of bacteria and their absence in humans, ECF transporters are novel potential antimicrobial targets to tackle infection. The hit compound’s metabolic and plasma stability, the potency (20, MIC Streptococcus pneumoniae = 2 µg/mL), the absence of cytotoxicity and a lack of resistance development under the conditions tested here suggest that this scaffold may represent a promising starting point for the development of novel antimicrobial agents with an unprecedented mechanism of action.<br>

scholarly journals Growth Promotion or Osmotic Stress Response: How SNF1-Related Protein Kinase 2 (SnRK2) Kinases Are Activated and Manage Intracellular Signaling in Plants

Plants ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 1443
Author(s):  
Yoshiaki Kamiyama ◽  
Sotaro Katagiri ◽  
Taishi Umezawa
Keyword(s):  
Protein Kinase ◽  
Plant Growth ◽  
Osmotic Stress ◽  
Protein Function ◽  
Intracellular Signaling ◽  
Growth Promotion ◽  
Research Field ◽  
Dependent Manner ◽  
Related Protein ◽  
Wide Range

Reversible phosphorylation is a major mechanism for regulating protein function and controls a wide range of cellular functions including responses to external stimuli. The plant-specific SNF1-related protein kinase 2s (SnRK2s) function as central regulators of plant growth and development, as well as tolerance to multiple abiotic stresses. Although the activity of SnRK2s is tightly regulated in a phytohormone abscisic acid (ABA)-dependent manner, recent investigations have revealed that SnRK2s can be activated by group B Raf-like protein kinases independently of ABA. Furthermore, evidence is accumulating that SnRK2s modulate plant growth through regulation of target of rapamycin (TOR) signaling. Here, we summarize recent advances in knowledge of how SnRK2s mediate plant growth and osmotic stress signaling and discuss future challenges in this research field.

scholarly journals Consumer Neuroscience Techniques in Advertising Research: A Bibliometric Citation Analysis

2021 ◽  
Vol 13 (3) ◽  
pp. 1589
Author(s):  
Juan Sánchez-Fernández ◽  
Luis-Alberto Casado-Aranda ◽  
Ana-Belén Bastidas-Manzano
Keyword(s):  
Self Report ◽  
Future Research ◽  
Complex Nature ◽  
Clear Understanding ◽  
Continuous Growth ◽  
Main Research ◽  
Consumer Neuroscience ◽  
Starting Point ◽  
Wide Range ◽  
Performance Analysis Tools

The limitations of self-report techniques (i.e., questionnaires or surveys) in measuring consumer response to advertising stimuli have necessitated more objective and accurate tools from the fields of neuroscience and psychology for the study of consumer behavior, resulting in the creation of consumer neuroscience. This recent marketing sub-field stems from a wide range of disciplines and applies multiple types of techniques to diverse advertising subdomains (e.g., advertising constructs, media elements, or prediction strategies). Due to its complex nature and continuous growth, this area of research calls for a clear understanding of its evolution, current scope, and potential domains in the field of advertising. Thus, this current research is among the first to apply a bibliometric approach to clarify the main research streams analyzing advertising persuasion using neuroimaging. Particularly, this paper combines a comprehensive review with performance analysis tools of 203 papers published between 1986 and 2019 in outlets indexed by the ISI Web of Science database. Our findings describe the research tools, journals, and themes that are worth considering in future research. The current study also provides an agenda for future research and therefore constitutes a starting point for advertising academics and professionals intending to use neuroimaging techniques.

Energy-minimization multiscale based mesoscale modeling and applications in gas-fluidized catalytic reactors

2019 ◽  
Vol 35 (8) ◽  
pp. 879-915 ◽  
Author(s):  
Bona Lu ◽  
Yan Niu ◽  
Feiguo Chen ◽  
Nouman Ahmad ◽  
Wei Wang ◽  
...  
Keyword(s):  
Energy Minimization ◽  
Fluid Model ◽  
Scale Up ◽  
Flow Characteristics ◽  
Chemical Kinetic ◽  
Mesoscale Modeling ◽  
Catalytic Reactors ◽  
Chemical Kinetic Model ◽  
Starting Point ◽  
Wide Range

Abstract Gas-solid fluidization is intrinsically dynamic and manifests mesoscale structures spanning a wide range of length and timescales. When involved with reactions, more complex phenomena emerge and thus pose bigger challenges for modeling. As the mesoscale is critical to understand multiphase reactive flows, which the conventional two-fluid model without mesoscale modeling may be inadequate to resolve even using extremely fine grids, this review attempts to demonstrate that the energy-minimization multiscale (EMMS) model could be a starting point to develop such mesoscale modeling. Then, the EMMS-based mesoscale modeling with emphasis on formulation of drag coefficients for different fluidization regimes, modification of mass transfer coefficient, and other extensions are discussed in an attempt to resolve the emerging challenges. Its applications with examples of development of novel fluid catalytic cracking and methanol-to-olefins processes prove that the mesoscale modeling plays a remarkable role in improving the predictions in hydrodynamic behaviors and overall reaction rate. However, the product content primarily depends on the chemical kinetic model itself, suggesting the necessity of an effective coupling between chemical kinetics and flow characteristics. The mesoscale modeling can be believed to accelerate the traditional experimental-based scale-up process with much lower cost in the future.

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