A cost-effective detection of low-abundance mutation with DNA three-way junction structure and lambda exonuclease

Feng, Zishan; Zhang, Wei; Li, Longjie; Tu, Bocheng; Ye, Weicong; Tang, Xiaofeng; Wang, Hongbo; Xiao, Xianjin; Wu, Tongbo

doi:10.1016/j.cclet.2020.06.009

Cited by 8 publications

(3 citation statements)

References 27 publications

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“…Many enzymes, such as Endonuclease IV (Endo IV), Lambda exonuclease and ligase, have extra selectivity to the matched and mismatched DNA base pairs. [ 10‐16 ] Thus, these enzymes would provide additional discrimination ability. They could also produce signal amplification through target cycling.…”

Section: Background and Originality Contentmentioning

confidence: 99%

Sensitive DNA Mutation Detection at Physiological Temperature with Endonuclease IV by Inhibiting Its Side Activity

Zhang

Liu

et al. 2021

Chin. J. Chem.

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Main observation and conclusion Endonuclease IV (Endo IV) has the side activity to cleave the apurinic/apyrimidinic site in the single‐stranded DNA probe (AP‐ssDNA), which adversely affects its performance in single‐nucleotide variation (SNV) detection. In this work, we developed a simple strategy to inhibit Endo IV's side activity by introducing an assistant strand into the detection system. The assistant strand effectively inhibited the side‐activity of Endo IV by confining the freedom of AP‐ssDNA through rigid double‐stranded DNA formation, and the inhibition efficiency could reach 98%. About 20 times enhancement of the discrimination factor (DF) was obtained compared to the detection system without the assistant strand. After optimization, a mean DF value was calculated to be 738 for different mutation types, and the sample with 0.005% allele frequency was discriminated from the wild‐type target. Another advantage of using an assistant strand was that the SNV detection could be executed at physiological temperature without precise temperature optimization as in other Endo IV‐based SNV detection system. Besides, EGFR T790M mutant in the synthesized and clinical samples was detected by our strategy. The results showed satisfactory sensitivity and accuracy by comparison with Sanger sequencing. Thus, this strategy has the potential to be applied in the field of precision medicine.

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Section: Background and Originality Contentmentioning

confidence: 99%

Sensitive DNA Mutation Detection at Physiological Temperature with Endonuclease IV by Inhibiting Its Side Activity

Zhang

Liu

et al. 2021

Chin. J. Chem.

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“…The selective recognition of short oligonucleotides, such as antisense oligo-DNA and miRNAs, at the single-molecule level is a critical area of research as it enables the development of early cancer diagnosis and drug development. − Nanopore sensing provides an innovative platform for single-molecule characterization of oligonucleotides through monitoring the translocation and analyzing the distinct temporary changes in the ionic current . Solid-state nanopores are considered to be more stable and controllable than biological nanopores, which are often fragile and difficult to manipulate. − This is due to the fact that solid-state nanopores are fabricated using diverse manufacturing techniques, which allows for precise control over their size, shape, and properties, − such as dielectric breakdown (CBD), transmission electron microscopy (TEM) beam, and ion beam irradiation .…”

Section: Introductionmentioning

confidence: 99%

Recognition of Oligonucleotide C by Polydopamine-Coated Solid-State Nanopores

Yang,

Yin,

Chen

et al. 2023

Anal. Chem.

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Selective recognition of short oligonucleotides at the single-molecule level is particularly important for early disease detection and treatment. In this work, polydopamine (PDA)-coated nanopores were prepared via self-polymerization as a solid-state nanopore sensing platform for the recognition of oligonucleotide C (PolyC). The PDA coating possesses abundant active sites, such as indole, amino, carboxyl, catechol, and quinone structures, which had interactions with short oligonucleotides to slow down the translocation rate. PDA-coated nanopores selectively interact with PolyC20 by virtue of differences in hydrogen bonding forces, generating a larger blocking current, while polyA and polyT demonstrated very small blockings. At the same time, PDA-coated nanopores can sensitively distinguish PolyC with different lengths, such as 20, 14, and 10 nt. The functionalization of PDA on the solid-state nanopore provides an opportunity for the rational design of the recognition surface for biomolecules.

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“…By tuning the length of the bulge loop, they realized the discrimination of SNV with a detection limit of 0.02% (MT abundance). Another way is introducing enzymes (polymerase, ligase, exonuclease, − endonuclease, , etc.) into the reaction system.…”

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confidence: 99%

Endonuclease IV-Regulated DNAzyme Motor for Universal Single-nucleotide Variation Discrimination

Zhang

Yuan

et al. 2021

Anal. Chem.

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Single-nucleotide variation (SNV) detection plays significant roles in disease diagnosis and treatment. Generally, auxiliary probe, restricted design rules, complicated detection system, and repeated experimental parameter optimization are needed to obtain satisfactory tradeoff between sensitivity and selectivity for SNV discrimination, especially when different mutant sites need to be distinguished. To overcome these limitations, we developed a universal, straightforward, and relatively cheap SNV discrimination strategy, which simultaneously possessed high sensitivity and selectivity. The excellent performance of this strategy was ascribed to the SNV discrimination property of endonuclease IV (Endo IV) and the different hydrolysis behavior between free deoxyribozyme (DNAzyme) and the trapped DNAzyme to the substrates modified on gold nanoparticles (AuNPs). When Endo IV recognized the mutant-type target (MT), free DNAzyme was released from the probe, and the DNAzyme motor was activated with the help of cofactor Mn2+ to generate an amplified fluorescence signal. On the contrary, the wild-type target (WT) could not effectively trigger the DNAzyme motor. Moreover, for different SNV types, the corresponding probe could be designed by simply changing the sequence hybridized with the target and retaining the DNAzyme sequence. Thus, the fluorescence signal generation system does not need to change for different SNV targets. Five clinical-related SNVs were determined with the limit of detection (LOD) ranging from 0.01 to 0.05%, which exhibited competitive sensitivity over existing SNV detection methods. This strategy provided another insight into the properties of Endo IV and DNAzyme, expanded the applications of DNAzyme motor, and has great potential to be used for precision medicine.

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A cost-effective detection of low-abundance mutation with DNA three-way junction structure and lambda exonuclease

Cited by 8 publications

References 27 publications

Sensitive DNA Mutation Detection at Physiological Temperature with Endonuclease IV by Inhibiting Its Side Activity

Sensitive DNA Mutation Detection at Physiological Temperature with Endonuclease IV by Inhibiting Its Side Activity

Recognition of Oligonucleotide C by Polydopamine-Coated Solid-State Nanopores

Endonuclease IV-Regulated DNAzyme Motor for Universal Single-nucleotide Variation Discrimination

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