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

Iserte, Javier Alonso; Stephan, Betina Inés; Goñi, Sandra Elizabeth; Borio, Cristina Silvia; Ghiringhelli, Pablo Daniel; Lozano, Mario Enrique

doi:10.1155/2013/383646

Cited by 29 publications

(23 citation statements)

References 30 publications

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“…Genomic knowledge of allergens also helps to define their common properties and will be helping to clear possible factors that cause allergenic potential in the future [37]. Basis necessity in degenerate primer designing is an alignment of selected nucleotide sequences [38]. Here, Bet v 1 was used as a model to analyze functionality of degenerate primers in clinically relevant cross species screening of genomic sequences of allergens ( Table 5).…”

Section: Technical Approaches and Methodologies For Pcr Screening Of mentioning

confidence: 99%

“…The design of degenerate primers for optimal PCR amplification should be based on a conserved region with a length of approximately 200-500 base pairs [38] what is the length that was positively identified in the screened Bet v 1 homologs. Degenerate primer is a mixture of oligonucleotide sequences, each of which has a slightly different sequence, that is, there are several probable bases in it.…”

Section: Technical Approaches and Methodologies For Pcr Screening Of mentioning

confidence: 99%

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Application of Genomic Data for PCR Screening of Bet v 1 Conserved Sequence in Clinically Relevant Plant Species

Žiarovská¹,

Zeleňáková²

2019

Systems Biology

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Bet v 1 is a highly immunogenic protein, which is the main cause of sensitivity to birch pollen and is described as the main birch allergen. Despite the structural similarity, Bet v 1 homologs show different properties and immunoreactivity. Here, the bioinformatic algorithms were applied for known Bet v 1 homologous nucleic acids sequences to find homology and conserved regions. Genomic sequences of PR proteins of two different fruit species, which allergens belong to PR proteins of the same type as Bet v 1, were selected to design degenerate primers. Subsequently, screening of the presence of Bet v 1 conserved genomic sequence was performed in 45 clinically relevant plant species.

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Section: Technical Approaches and Methodologies For Pcr Screening Of mentioning

confidence: 99%

Section: Technical Approaches and Methodologies For Pcr Screening Of mentioning

confidence: 99%

Application of Genomic Data for PCR Screening of Bet v 1 Conserved Sequence in Clinically Relevant Plant Species

Žiarovská¹,

Zeleňáková²

2019

Systems Biology

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“…DinoKnot was used with the RNA transcript region and the reverse primer to determine the location where the reverse primer binds and the secondary interaction structure. Two of the reverse primers, HKU-ORF1-R and RdRp-SARS-R, contain degenerate bases which means there are a mixture of oligonucleotides that contain different bases at the degenerate base position [31]. In these cases, all possible degenerate base combinations were predicted with DinoKnot.…”

Section: System and Experiments Setupmentioning

confidence: 99%

In silico prediction of COVID-19 test efficiency with DinoKnot

Newman

Chang

Jabbari

2020

Preprint

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The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel coronavirus spreading across the world causing the disease COVID-19. The diagnosis of COVID-19 is done by quantitative reverse-transcription polymer chain reaction (qRT-PCR) testing which utilizes different primer-probe sets depending on the assay used. Using in silico analysis we aimed to determine how the secondary structure of the SARS-CoV-2 RNA genome affects the interaction between the reverse primer during qRT-PCR and how it relates to the experimental primer-probe test efficiencies. We introduce the program DinoKnot (Duplex Interaction of Nucleic acids with pseudoKnots) that follows the hierarchical folding hypothesis to predict the secondary structure of two interacting nucleic acid strands (DNA/RNA) of similar or different type. DinoKnot is the first program that utilizes stable stems in both strands as a guide to find the structure of their interaction. Using DinoKnot we predicted the interaction of the reverse primers used in four common COVID-19 qRT-PCR tests with the SARS-CoV-2 RNA genome. In addition, we predicted how 12 mutations in the primer/probe binding region may affect the primer/probe ability and subsequent SARS-CoV-2 detection. While we found all reverse primers are capable of interacting with their target area, we identified partial mismatching between the SARS-CoV-2 genome and some reverse primers. We predicted three mutations that may prevent primer binding, reducing the ability for SARS-CoV-2 detection. We believe our contributions can aid in the design of a more sensitive SARS-CoV-2 test.Author summaryThe current testing for the disease COVID-19 that is caused by the novel cornonavirus SARS-CoV-2 uses oligonucleotides called primers that bind to specific target regions on the SARS-CoV-2 genome to detect the virus. Our goal was to use computational tools to predict how the structure of the SARS-CoV-2 RNA genome affects the ability of the primers to bind to their target region. We introduce the program DinoKnot (Duplex interaction of nucleic acids with pseudoknots) that is able to predict the interactions between two DNA or RNA molecules. We used DinoKnot to predict the efficiency of four common COVID-19 tests, and the effect of mutations in the SARS-CoV-2 virus on ability of the COVID-19 tests in detecting those strains. We predict partial mismatching between some primers and the SARS-CoV-2 genome but that all primers are capable of interacting with their target areas. We also predict three mutations that prevent primer binding and thus SARS-CoV-2 detection. We discuss the limitations of the current COVID-19 testing and suggest the design of a more sensitive COVID-19 test that can be aided by our findings.

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“…Previous applications of genetics‐based approaches have either used specific primers to search samples of fish eggs for species of interest (Chow & Inoue, ) or used degenerate primers and DNA barcoding to identify individual fish eggs to species level (Ivanova, Zemlak, Hanner, & Hebert, ). Degenerate primers are mixtures of oligonucleotide sequences (primers) that produce a larger range of possible nucleotide matches during PCR amplification (Iserte et al., ). Studies that have compared visual identifications of percomorph eggs with identifications obtained through DNA barcoding have indicated visual identifications can be highly unreliable (Larson et al., ).…”

Section: Introductionmentioning

confidence: 99%

DNA barcoding reveals clear delineation between spawning sites for neritic versus oceanic fishes in the Gulf of Mexico

Burrows

Browning

Breitbart

et al. 2018

Fisheries Oceanography

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We combined research‐vessel cruises of opportunity with DNA barcoding to survey planktonic, percomorph fish eggs at 40 stations distributed across and around the Gulf of Mexico (GoM). The objectives were (a) to determine whether eggs of fishes that are potential candidates for the daily egg production method (DEPM) can be readily barcoded, (b) to identify taxa that are spawning in the GoM, (c) to determine encounter rates for eggs of economically valuable taxa, and (d) to characterize individual egg taxa as being primarily neritic, primarily oceanic, or primarily mixed (i.e., both neritic and oceanic). Of the 1,144 eggs that were individually barcoded, 709 (62%) were definitively identified to species (62 species from 42 families), with an additional 20 taxa identified to genus or subfamily level. The eggs of 15 economically important species were identified, most of which had intermediate encounter rates and moderately dispersed spatial distributions, as indicated by an index of aggregation. SIMPROF analysis of stationwise cluster analysis identified eight significant groups within the 35 stations that yielded percomorph eggs; a corresponding specieswise analysis identified six groups of stations as having a neritic egg community and two groups as having an oceanic community, with a community transition located at the shelf break. Although the neritic and oceanic stations did not share important species, it remains possible that coastal pelagic species have mixed neritic–oceanic distributions. Together, these results indicate DEPM fish‐egg surveys based on DNA barcoding are feasible at the large marine ecosystem scale.

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

Cited by 29 publications

References 30 publications

Application of Genomic Data for PCR Screening of Bet v 1 Conserved Sequence in Clinically Relevant Plant Species

Application of Genomic Data for PCR Screening of Bet v 1 Conserved Sequence in Clinically Relevant Plant Species

In silico prediction of COVID-19 test efficiency with DinoKnot

DNA barcoding reveals clear delineation between spawning sites for neritic versus oceanic fishes in the Gulf of Mexico

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