Sensitive discrimination of stable mismatched base pairs by an abasic site modified fluorescent probe and lambda exonuclease

Wu, Tongbo; Xiao, Xianjin; Gu, Feidan; Zhao, Meiping

doi:10.1039/c5cc05749c

Cited by 14 publications

(15 citation statements)

References 42 publications

(73 reference statements)

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“…They can also be used to detect mutations at the 3′ end of a target sequence with high DFs. In comparison with those previously reported fluorescent probes designed for post-PCR mutation detection ( 44 , 51–54 ), a distinct advantage of the 5′-overhang probe was the ultra-high sensitivity to low-abundance mutations, which should be attributed to the special interactions between λ exo and dsDNA with a 5′-FAM 2-nt overhang terminal structure. This unique structure brought the enzyme an extraordinary discrimination capability toward the presence of mismatched base pairs in the duplex region near the 5′ end, thus the DFs between MT and WT are very high.…”

Section: Resultsmentioning

confidence: 86%

“…For EGFR G719S (NM_005228.4:c.2155G>A), the mismatched base pair formed at Position 4 in the probe/WT hybrid was a stable T:G mismatch, which was relatively hard to be discriminated. So we introduced a blocker which had the same length as the probe and perfectly matched with the WT sequence to suppress the background signals ( 44 ). As the probe had two mismatches with WT, the blocker would significantly impede the hybridization of probe and WT.…”

Section: Resultsmentioning

confidence: 99%

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DNA terminal structure-mediated enzymatic reaction for ultra-sensitive discrimination of single nucleotide variations in circulating cell-free DNA

Chen

Yang

et al. 2017

Nucleic Acids Research

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Sensitive detection of the single nucleotide variants in cell-free DNA (cfDNA) may provide great opportunity for minimally invasive diagnosis and prognosis of cancer and other related diseases. Here, we demonstrate a facile new strategy for quantitative measurement of cfDNA mutations at low abundance in the cancer patients’ plasma samples. The method takes advantage of a novel property of lambda exonuclease which effectively digests a 5′-fluorophore modified dsDNA with a 2-nt overhang structure and sensitively responds to the presence of mismatched base pairs in the duplex. It achieves a limit of detection as low as 0.02% (percentage of the mutant type) for BRAFV600E mutation, NRASQ61R mutation and three types of EGFR mutations (G719S, T790M and L858R). The method enabled identification of BRAFV600E and EGFRL858R mutations in the plasma of different cancer patients within only 3.5 h. Moreover, the terminal structure-dependent reaction greatly simplifies the probe design and reduces the cost, and the assay only requires a regular real-time PCR machine. This new method may serve as a practical tool for quantitative measurement of low-abundance mutations in clinical samples for providing genetic mutation information with prognostic or therapeutic implications.

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Section: Resultsmentioning

confidence: 86%

Section: Resultsmentioning

confidence: 99%

DNA terminal structure-mediated enzymatic reaction for ultra-sensitive discrimination of single nucleotide variations in circulating cell-free DNA

Chen

Yang

et al. 2017

Nucleic Acids Research

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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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“… 37 – 42 However, most of these efforts have focused on the optimization of reaction condition and probe or primer design, and the discrimination ability is still seriously limited by the cross-reactivity with closely related unintended sequences. Recently, several competitive systems using the sequence-specific DNA sinks, 3 , 43 the controller DNAs, 44 the DNA-blocker strand 45 and the peptide nucleic acid clamps 46 were designed for SNM discrimination. These advances effectively improved the discrimination ability but suffered from complexity and required stringent condition control.…”

Section: Introductionmentioning

confidence: 99%

Ultra-specific discrimination of single-nucleotide mutations using sequestration-assisted molecular beacons

Tang

Zhao

et al. 2017

Chem. Sci.

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Sensitive discrimination of stable mismatched base pairs by an abasic site modified fluorescent probe and lambda exonuclease

Cited by 14 publications

References 42 publications

DNA terminal structure-mediated enzymatic reaction for ultra-sensitive discrimination of single nucleotide variations in circulating cell-free DNA

DNA terminal structure-mediated enzymatic reaction for ultra-sensitive discrimination of single nucleotide variations in circulating cell-free DNA

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

Ultra-specific discrimination of single-nucleotide mutations using sequestration-assisted molecular beacons

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