PubMed · 8650348
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Y Murakami. 1996. [Reverse transcriptase-polymerase chain reaction].. https://pubmed.ncbi.nlm.nih.gov/8650348/
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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.
Here we describe template directed enzymatic synthesis of unique primers, avoiding the chemical synthesis step in primer walking. We have termed this conceptually new technique DENS (differential extension with nucleotide subsets). DENS works by selectively extending a short primer, making it a long one at the intended site only. The procedure starts with a limited initial extension of the primer (at 20-30 degrees C) in the presence of only two out of the four possible dNTPs. The primer is extended by 6-9 bases or longer at the intended priming site, which is deliberately selected, (as is the two-dNTP set), to maximize the extension length. The subsequent termination reaction at 60-65 degrees C then accepts the extended primer at the intended site, but not at alternative sites, where the initial extension (if any) is generally much shorter. DENS allows the use of primers as long as 8mers (degenerate in two positions) which prime much more strongly than modular primers involving 5-7mers and which (unlike the latter) can be used with thermostable polymerases, thus allowing cycle-sequencing with dye-terminators compatible with Taq DNA polymerase, as well as making double-stranded DNA sequencing more robust.
We investigated the effects of internal primer-template mismatches on the efficiency of reverse transcription and PCR amplification. As models, RNA transcripts representative of different HIV-1 group M subtypes were evaluated with a previously described gag primer pair system. We observed that the presence of two to four mismatches in the primer-template duplexes did not have a significant effect on RT-PCR. However, the presence of five and six mismatches with the 28 and 30 base primers reduced PCR product yield by approximately 22- and 100-fold respectively, relative to the homologous template. The amount of reduction was reproducible from experiment to experiment and was independent of the initial copy number input. Under the conditions used, viral RNA measurements of the more divergent HIV-1 subtypes (A and E) would be underestimated, while isolates of subtypes B, C, D and F-H are expected to be efficiently amplified and accurately measured. The reduced amplification efficiency for targets similar to HIV subtypes A and E can be improved 4- to 10-fold by lowering the annealing temperature and implementing a reverse transcription step that gradually increases in temperature. The additional substitution of either 5-methylcytosine for cytosine throughout or the substitution of inosine at positions of variable bases resulted in a <4-fold difference in product yield between the homologous and most divergent templates.