Fluorogenic oligonucleotide cleavage probes with a branched linker improve RNA detection
Bibliographic record
Abstract
Fluorescent probe-based quantitative polymerase chain reaction (qPCR) is essential for DNA/RNA quantification widely used in research and clinical diagnostics. The performance of fluorogenic probes depends heavily on their design, particularly the identities of the fluorophore and quencher moieties, and the linkers used to attach them to oligonucleotides. Here we report a highly modular, three-way branched glycerol 'X' linker in fluorogenic TaqMan® type oligonucleotide probes for multiplexed, reverse transcription qPCR (RT-qPCR). The flexible 'X' linker served as an internal attachment point for various quenchers (BHQ1, BHQ2) in probes containing a variable fluorophore at the 5' end (Flu, Hex, Cy5, Cy5.5). A four-color RT-qPCR 'tetraplex' assay was thereby developed for distinguishing between RNA genomes from SARS-CoV-2, influenza A, and influenza B viruses in a single reaction. The 'X' linker exhibited superior performance with single-molecule detection limits approaching four copies, compared to an internal arabinoside-based (ara) linker strategy, demonstrating the presence of competing processes during primer extension, one where Taq exonuclease activity cleaves the fluorogenic X probe leading to productive fluorescence, and the second where the ara probe is displaced from the PCR template without cleavage. Together these results demonstrate the importance of linker structure selection in oligonucleotides for developing highly effective fluorogenic probes for qPCR.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.001 | 0.001 |
| Meta-epidemiology (narrow) | 0.001 | 0.001 |
| Meta-epidemiology (broad) | 0.001 | 0.001 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.001 |
| Scholarly communication | 0.001 | 0.001 |
| Open science | 0.001 | 0.001 |
| Research integrity | 0.001 | 0.001 |
| Insufficient payload (model declined to judge) | 0.002 | 0.002 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".