Accurate genotyping of fragmented DNA using a toehold assisted padlock probe

Gao, Yanmin; Qiao, Hongyan; Pan, Victor Y.; Wang, Zhaoguan; Li, Jiaojiao; Wei, Yanan; Ke, Yonggang; Qi, Hao

doi:10.1016/j.bios.2021.113079

Cited by 12 publications

(4 citation statements)

References 26 publications

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“…The same idea may be applicable to other biotech-nology such as genome editing 24 , DNA nanotechnologies 25 , and RNA interference 26 , which also rely on specific hybridization of nucleic acids. In fact, a similar method that blocks unnecessary binding has been recently reported for DNA ligation 27 .…”

Section: Discussionmentioning

confidence: 96%

Error-suppression mechanism of PCR by blocker strands

Aoyanagi

Pigolotti

Ono

et al. 2022

Preprint

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The polymerase chain reaction (PCR) is a central technique in biotechnology. Its ability to amplify a specific target region of a DNA sequence has led to prominent applications, including virus tests, DNA sequencing, genotyping, and genome cloning. These applications rely on the specificity of the primer hybridization, and therefore require effective suppression of hybridization errors. This suppression is usually based on the energetic stability of correct hybridization. The performance of this traditional approach requires a careful design of the primer sequence and a high annealing temperature and has inherent limitations, for example in terms of reaction efficiency. Here we show that, by adding a "blocker strand" to the PCR mixture, we can sculpt a kinetic barrier that complements the traditional energetic biasing. Our method drastically suppresses the replication error by PCR without compromising the reaction efficiency. It also extends the viable range of annealing temperatures and reduces design constraint of the primer sequence. Thanks to these properties, we expect our method to significantly broaden and improve the applicability of PCR. Our approach may be extended to other biotechnology including genome editing, DNA nanotechnologies, and RNA interference.

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Section: Discussionmentioning

confidence: 96%

Error-suppression mechanism of PCR by blocker strands

Aoyanagi

Pigolotti

Ono

et al. 2022

Preprint

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“…For instance, the target recognition process only depends on base complementary pairing and the fidelity of the utilized ligase to ensure specificity, which is insufficient for distinguishing single-base mismatch. 14 Additionally, nonspecific cyclization and intermolecular crossreaction can occur, 15 which significantly reduces the signalto-noise ratio (SNR) and limits the detection sensitivity.…”

Section: Introductionmentioning

confidence: 99%

A Universal Strategy for Enhancing the Circulating miRNAs’ Detection Performance of Rolling Circle Amplification by Using a Dual-Terminal Stem-Loop Padlock

Xu,

Wu,

Liu

et al. 2023

ACS Nano

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Rolling circle amplification (RCA) is one of the most promising nucleic acid detection technologies and has been widely used in the molecular diagnosis of disease. Padlock probes are often used to form circular templates, which are the core of RCA. However, RCA often suffers from insufficient specificity and sensitivity. Here we report a reconstruction strategy for conventional padlock probes to promote their overall performance in nucleic acid detection while maintaining probe functions uncompromised. When two rationally designed stem-loops were strategically placed at the two terminals of linear padlock probes, the specificity of target recognition was enhanced and the negative signal was significantly delayed. Our design achieved the best single-base discrimination compared with other structures and over a 1000-fold higher sensitivity than that of the conventional padlock probe, validating the effectiveness of this reconstruction. In addition, the underlying mechanisms of our design were elucidated through molecular dynamics simulations, and the versatility was validated with longer and shorter padlocks targeting the same target, as well as five additional targets (four miRNAs and dengue virus – 2 RNA mimic (DENV-2)). Finally, clinical applicability in multiplex detection was demonstrated by testing real plasma samples. Our exploration of the structures of nucleic acids provided another perspective for developing high-performance detection systems, improving the efficacy of practical detection strategies, and advancing clinical diagnostic research.

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“…Cell-free DNA (cfDNA), a typical fragmented DNA, as a noninvasive diagnostic biomarker has aroused great interest . In addition, cfDNA plays a vital role in biomedical diagnosis, such as early diagnosis of cancer, assessment of tumor stage, selection of effective therapeutic schemes, and monitoring prognosis .…”

mentioning

confidence: 99%

“…C ell-free DNA (cfDNA), a typical fragmented DNA, as a noninvasive diagnostic biomarker has aroused great interest. 1 In addition, cfDNA plays a vital role in biomedical diagnosis, 2 such as early diagnosis of cancer, 3 assessment of tumor stage, 4 selection of effective therapeutic schemes, 5 and monitoring prognosis. 6 Polymerase chain reaction (PCR)based methods such as droplet digital PCR (ddPCR), 7 asymmetric PCR, 8 emulsion digital PCR, 9 and reverse transcription quantitative PCR (RT-qPCR) 10 are considered as gold standards for cfDNA assay.…”

mentioning

confidence: 99%

Smartphone-Based Pure DNAzyme Hydrogel Platform for Visible and Portable Colorimetric Detection of Cell-Free DNA

et al. 2022

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Cell-free DNA (cfDNA), as a tumor marker, is of great importance for the diagnosis of cancer and targeted therapy. However, the need for huge analytical instruments for cfDNA analysis has restricted its practical applications, especially in rural areas and third-world countries. Herein, a portable and visual smartphone-based DNAzyme hydrogel platform is developed for cfDNA detection. The target cfDNA triggers rolling circle amplification to produce a G-quadruplex-comprised DNA hydrogel with an horseradish peroxidase (HRP)-like catalytic function, which further catalyzes the chromogenic substrate to generate a visible output signal. Notably, the naked-eye detection of cfDNA can be realized by the macroscale visibility and catalytic ability of the DNA hydrogel. The linear range of the DNAzyme hydrogel platform for cfDNA detection is 0.1 pM−1500 nM with a detection limit of 0.042 pM. Moreover, this platform is exploited for the detection of cfDNA in spiked human serum with favorable sensitivity and recovery. Therefore, the DNAzyme hydrogel platform provides highly promising potential for testing other nucleic acid biomarkers.

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Accurate genotyping of fragmented DNA using a toehold assisted padlock probe

Cited by 12 publications

References 26 publications

Error-suppression mechanism of PCR by blocker strands

Error-suppression mechanism of PCR by blocker strands

A Universal Strategy for Enhancing the Circulating miRNAs’ Detection Performance of Rolling Circle Amplification by Using a Dual-Terminal Stem-Loop Padlock

Smartphone-Based Pure DNAzyme Hydrogel Platform for Visible and Portable Colorimetric Detection of Cell-Free DNA

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