scholarly journals Impact of metal oxide nanoparticles on in vitro DNA amplification

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e7228 ◽  
Author(s):  
Chun-Hui Gao ◽  
Monika Mortimer ◽  
Ming Zhang ◽  
Patricia A. Holden ◽  
Peng Cai ◽  
...  
Keyword(s):  
Metal Oxide ◽  
Dna Polymerase ◽  
Metal Oxide Nanoparticles ◽  
Pcr Amplification ◽  
Dna Amplification ◽  
Amplicon Sequencing ◽  
High Fidelity ◽  
Nucleotide Polymorphisms ◽  
Single Nucleotide

Polymerase chain reaction (PCR) is used as an in vitro model system of DNA replication to assess the genotoxicity of nanoparticles (NPs). Prior results showed that several types of NPs inhibited PCR efficiency and increased amplicon error frequency. In this study, we examined the effects of various metal oxide NPs on inhibiting PCR, using high- vs. low-fidelity DNA polymerases; we also examined NP-induced DNA mutation bias at the single nucleotide level. The effects of seven major types of metal oxide NPs (Fe2O3, ZnO, CeO2, Fe3O4, Al2O3, CuO, and TiO2) on PCR replication via a low-fidelity DNA polymerase (Ex Taq) and a high-fidelity DNA polymerase (Phusion) were tested. The successfully amplified PCR products were subsequently sequenced using high-throughput amplicon sequencing. Using consistent proportions of NPs and DNA, we found that the effects of NPs on PCR yield differed depending on the DNA polymerase. Specifically, the efficiency of the high-fidelity DNA polymerase (Phusion) was significantly inhibited by NPs during PCR; such inhibition was not evident in reactions with Ex Taq. Amplicon sequencing showed that the overall error rate of NP-amended PCR was not significantly different from that of PCR without NPs (p > 0.05), and NPs did not introduce single nucleotide polymorphisms during PCR. Thus, overall, NPs inhibited PCR amplification in a DNA polymerase-specific manner, but mutations were not introduced in the process.

scholarly journals Somatic variants for seed and fruit set in grapevine

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Laura Costantini ◽  
Paula Moreno-Sanz ◽  
Chinedu Charles Nwafor ◽  
Silvia Lorenzi ◽  
Annarita Marrano ◽  
...  
Keyword(s):  
Single Nucleotide Polymorphisms ◽  
Fruit Set ◽  
Climatic Factors ◽  
Embryo Sac ◽  
Wine Industry ◽  
Nucleotide Polymorphisms ◽  
Wine Quality ◽  
Single Nucleotide ◽  
Germplasm Collections

Abstract Background Grapevine reproductive development has direct implications on yield. It also impacts on berry and wine quality by affecting traits like seedlessness, berry and bunch size, cluster compactness and berry skin to pulp ratio. Seasonal fluctuations in yield, fruit composition and wine attributes, which are largely driven by climatic factors, are major challenges for worldwide table grape and wine industry. Accordingly, a better understanding of reproductive processes such as gamete development, fertilization, seed and fruit set is of paramount relevance for managing yield and quality. With the aim of providing new insights into this field, we searched for clones with contrasting seed content in two germplasm collections. Results We identified eight variant pairs that seemingly differ only in seed-related characteristics while showing identical genotype when tested with the GrapeReSeq_Illumina_20K_SNP_chip and several microsatellites. We performed multi-year observations on seed and fruit set deriving from different pollination treatments, with special emphasis on the pair composed by Sangiovese and its seedless variant locally named Corinto Nero. The pollen of Corinto Nero failed to germinate in vitro and gave poor berry set when used to pollinate other varieties. Most berries from both open- and cross-pollinated Corinto Nero inflorescences did not contain seeds. The genetic analysis of seedlings derived from occasional Corinto Nero normal seeds revealed that the few Corinto Nero functional gametes are mostly unreduced. Moreover, three genotypes, including Sangiovese and Corinto Nero, were unexpectedly found to develop fruits without pollen contribution and occasionally showed normal-like seeds. Five missense single nucleotide polymorphisms were identified between Corinto Nero and Sangiovese from transcriptomic data. Conclusions Our observations allowed us to attribute a seedlessness type to some variants for which it was not documented in the literature. Interestingly, the VvAGL11 mutation responsible for Sultanina stenospermocarpy was also discovered in a seedless mutant of Gouais Blanc. We suggest that Corinto Nero parthenocarpy is driven by pollen and/or embryo sac defects, and both events likely arise from meiotic anomalies. The single nucleotide polymorphisms identified between Sangiovese and Corinto Nero are suitable for testing as traceability markers for propagated material and as functional candidates for the seedless phenotype.

In-Silico and In-vitro Analysis of Human SOS1 Protein Causing Noonan Syndrome – A Novel Approach to Explore the Molecular Pathways

2021 ◽  
Vol 22 ◽  
Author(s):  
Vinoth Sigamani ◽  
Sheeja Rajasingh ◽  
Narasimman Gurusamy ◽  
Arunima Panda ◽  
Johnson Rajasingh
Keyword(s):  
Noonan Syndrome ◽  
In Silico ◽  
Genetic Disorder ◽  
In Vitro Study ◽  
Human Skin Fibroblast ◽  
Nucleotide Polymorphisms ◽  
Gene Expressions ◽  
Single Nucleotide ◽  
Nucleotide Mutation

Aims: Noonan syndrome (NS) is an autosomal dominant genetic disorder caused by single nucleotide mutation in PTPN11, SOS1, RAF1, and KRAS genes. Background: We hypothesize that in-silico analysis of human SOS1 mutations would be a promising predictor in identifying the pathogenic effect of NS. Methods: Here, we computationally analyzed the SOS1 gene to identify the pathogenic non-synonymous single nucleotide polymorphisms (nsSNPs) to cause NS. The variant information of SOS1 was collected from the SNP database (dbSNP). The variants were further analyzed by in-silico tools I-Mutant, iPTREE-STAB, and MutPred to elucidate their structural and functional characteristics. Results: We found that 11 nsSNPs of SOS1 were more pathogenic to cause NS. The 3D modeling of the wild-type and the 11 nsSNPs were performed using I-TASSER and validated via ERRAT and RAMPAGE. SOS1 interacting proteins were analysed through STRING, which showed that SOS1 interacted with cardiac proteins GATA4, TNNT2, and ACTN2. During these interactions, GRB2 and HRAS act as an intermediate molecules between SOS1 and cardiac proteins. These in-silico analyses were validated using induced cardiomyocytes (iCMCs) derived from NS patients carrying SOS1 gene variant c.1654A>G (NS-iCMCs) and compared with control human skin fibroblast-derived iCMCs (C-iCMCs). Our in vitro data further confirmed that the SOS1, GRB2 and HRAS gene expressions as well as the activated ERK protein, were significantly decreased in NS-iCMCs compared to C-iCMCs. Conclusion: This is the first in-silico and in vitro study demonstrating that 11 nsSNPs of SOS1 were playing a deleterious pathogenic role in causing NS.

Contribution of boars to reproductive performance and paternity after homospermic and heterospermic artificial insemination

2015 ◽  
Vol 27 (7) ◽  
pp. 1012 ◽  
Author(s):  
C. E. R. Ferreira ◽  
D. B. Sávio ◽  
A. C. Guarise ◽  
M. J. Flach ◽  
G. D. A. Gastal ◽  
...  
Keyword(s):  
Reproductive Performance ◽  
Artificial Insemination ◽  
Penetration Rate ◽  
Nucleotide Polymorphisms ◽  
Single Nucleotide ◽  
Blood Samples ◽  
Precise Evaluation ◽  
Umbilical Cords ◽  
Farrowing Rate

Heterospermic AI is commonly used in swine despite preventing precise evaluation of individual boar fertility. The present study compared the contribution of four boars (A, B, C and D) for reproductive performance and for paternity using homospermic and heterospermic (AB, AC, AD, BC, BD and CD) AI (n = 204 for homospermic AI; n = 307 for heterospermic AI). Blood samples from the four boars, from all sows inseminated with heterospermic doses and from the umbilical cords of their piglets, as well as tissue smears from mummified fetuses, were genotyped using single nucleotide polymorphisms (SNPs). Differences among boars were detected for the in vitro oocyte penetration rate and for the number of spermatozoa per oocyte (P < 0.05), but not for sperm motility, mitochondrial functionality and integrity of the membrane, acrosome and DNA (P > 0.05). Homospermic and heterospermic AI resulted in similar (P > 0.05) farrowing rates (90.5% and 89.9%, respectively) and total litter size (12.4 ± 0.4 and 12.7 ± 0.7, respectively). Farrowing rate was lower for Boar B than for Boar C (P < 0.05), but no other differences in reproductive performance among boars were observed with homospermic AI. The SNPs determined the paternity of 94.2% of the piglets sired by heterospermic AI. In the AC pool, paternity contribution per boar was similar (P > 0.05), but differences between boars occurred in all other pools (P < 0.05). Boar D achieved the greatest paternity contribution in all pools and parity categories (nearly 60%), whereas Boar B sired the fewest piglets (at most 40%). Reproductive performance was similar with homospermic and heterospermic AI, but differences in performance among boars undetected with homospermic AI were only evident after genotyping the piglets sired through heterospermic AI.

scholarly journals Population structure of eulachon Thaleichthys pacificus from Northern California to Alaska using single nucleotide polymorphisms from direct amplicon sequencing

2020 ◽  
Author(s):  
Ben J. G. Sutherland ◽  
John Candy ◽  
Kayla Mohns ◽  
Olivia Cornies ◽  
Kim Jonsen ◽  
...  
Keyword(s):  
Gulf Of Alaska ◽  
Amplicon Sequencing ◽  
Fraser River ◽  
Northern California ◽  
Nucleotide Polymorphisms ◽  
Single Nucleotide ◽  
Southeast Alaska ◽  
Southern Us ◽  
Mixed Stock ◽  
Future Work

ABSTRACTEulachon Thaleichthys pacificus, a culturally and ecologically important anadromous smelt (Family Osmeridae), ranges from Northern California to the southeast Bering Sea. In recent decades, some populations have experienced declines. Here we use a contig-level genome assembly combined with previously published RADseq-derived markers to construct an amplicon panel for eulachon. Using this panel, we develop a filtered genetic baseline of 521 variant loci genotyped in 1,989 individuals from 14 populations ranging from Northern California through Central Alaska. Consistent with prior genetic studies, the strongest separation occurs among three main regions: from Northern California up to and including the Fraser River; north of the Fraser River to southeast Alaska; and within the Gulf of Alaska. Separating the Fraser River from southern US populations, and refining additional substructure within the central coast may be possible in mixed-stock analysis; this will be addressed in future work. The amplicon panel outperformed the previous microsatellite panel, and thus will be used in future mixed-stock analyses of eulachon in order to provide new insights for management and conservation of eulachon.

scholarly journals Single-Nucleotide-Polymorphism-Specific PCR for Quantification and Discrimination of Chlamydia pneumoniae Genotypes by Use of a “Locked” Nucleic Acid

2006 ◽  
Vol 72 (5) ◽  
pp. 3785-3787 ◽  
Author(s):  
Jan Rupp ◽  
Werner Solbach ◽  
Jens Gieffers
Keyword(s):  
Single Nucleotide Polymorphism ◽  
Chlamydia Pneumoniae ◽  
Locked Nucleic Acid ◽  
Nucleotide Polymorphisms ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Specific Pcr ◽  
Intraspecies Diversity

ABSTRACT Single-nucleotide polymorphisms (SNPs) are targets to discriminate intraspecies diversity of bacteria and to correlate a genotype with a potential pathotype. Quantification of polygenotypic populations supports this task for in vitro and in vivo applications. We present a novel assay capable of quantifying mixtures of two genotypes differing by only one SNP.

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