PubMed · 7739906
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G Sarkar, M E Bolander. 1995-04-11. Semi exponential cycle sequencing.. https://doi.org/10.1093/nar%2F23.7.1269
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Plant viruses pose a significant threat to global agriculture and require efficient tools for their timely detection. We present AutoPVPrimer, an innovative pipeline that integrates artificial intelligence (AI) and machine learning to accelerate the development of plant virus primers. The pipeline uses Biopython to automatically retrieve different genomic sequences from the NCBI database to increase the robustness of the subsequent primer design. The design_primers_with_tuning module uses a random forest classifier that optimizes parameters and provides flexibility for different experimental conditions. Quality control measures, including the evaluation of poly-X content and melting temperature, increase primer reliability. Unique to AutoPVPrimer is the visualize_primer_dimer module, which supports the visual evaluation of primer dimers-a feature missing in other tools. Primer specificity is validated via primer BLAST, which contributes to the overall efficiency of the pipeline. AutoPVPrimer has been successfully applied to the tomato mosaic virus, proving its adaptability and efficiency. The modular design allows customization by the user and extends the applicability to different plant viruses and experimental scenarios. The pipeline represents a significant advance in primer design and provides researchers with an effective tool to accelerate molecular biology experiments. Future developments aim to extend compatibility and incorporate user feedback to consolidate AutoPVPrimer as an innovative contribution to the bioinformatics toolbox and a promising resource for the advancement of plant virology research.
Genome heterogeneity in retroviruses derives from poor fidelity of the reverse transcriptase (RT) and recombination via RT-catalyzed strand transfer synthesis. RTs lack proofreading ability, and they proficiently extend primers with mismatched termini. Recombination reactions carried out in vitro are accompanied by a high frequency of base substitution errors, suggesting a relationship. Here we provide evidence that misincorporation during RNA-directed DNA synthesis promotes strand transfer recombination. Experiments involved measurement of DNA synthesis, RNase H-directed cleavage, and strand transfer synthesis from preformed mismatched primers on RNA templates by human immunodeficiency virus (HIV) RT in vitro. A significant pause in synthesis occurred from a G(primer). rA(template) mismatch compared to the synthesis from a correctly paired (T.A) primer. The misincorporation-induced pause allowed an unusually large area of RT-RNase H-directed cleavage of the template RNA beneath the primer. Strand transfer to an acceptor molecule with sequence identical to the template RNA was about 50% more efficient than if the primer had had a correctly paired terminus. Overall transfer was measured over a large region of homology. Assuming that enhanced transfer occurs primarily at the site of the mismatch, the actual increase in transfer at that site must have been 1-2 orders of magnitude. Inclusion of a different acceptor molecule with complete complementarity to the originally mismatched 3' primer terminus resulted in an additional 2-fold increase in strand transfer efficiency. Overall, these results suggest the mechanism by which misincorporation during minus strand DNA synthesis in retroviral replication would promote high frequency recombination.
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