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Sensitive DNA Mutation Detection at Physiological Temperature with Endonuclease IV by Inhibiting Its Side Activity
Author(s) -
Zhang Zhen,
Li Longjie,
Liu Qingxu,
Hu Yuqiang,
Yuan Wenqian,
Xiao Xianjin,
Wu Tongbo
Publication year - 2021
Publication title -
chinese journal of chemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.28
H-Index - 41
eISSN - 1614-7065
pISSN - 1001-604X
DOI - 10.1002/cjoc.202100349
Subject(s) - endonuclease , chemistry , sanger sequencing , ap site , dna , cleave , mutant , single strand , microbiology and biotechnology , mutation , mutagenesis , restriction enzyme , biophysics , biochemistry , dna sequencing , gene , biology
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.