TitleDetection of low-frequency mutations in clinical samples by increasing mutation abundance via the excision of wild-type sequences
AuthorsChen, Wei
Xu, Haiqi
Dai, Shenbin
Wang, Jiayu
Yang, Ziyu
Jin, Yuewen
Zou, Mengbing
Xiao, Xianjin
Wu, Tongbo
Yan, Wei
Zhang, Bin
Lin, Zhimiao
Zhao, Meiping
AffiliationPeking Univ, Coll Chem & Mol Engn, Beijing Natl Lab Mol Sci, Beijing, Peoples R China
Peking Univ, Coll Chem & Mol Engn, MOE Key Lab Bioorgan Chem & Mol Engn, Beijing, Peoples R China
Huazhong Univ Sci & Technol, Inst Reprod Hlth, Tongji Med Coll, Wuhan, Peoples R China
Huazhong Univ Sci & Technol, Tongji Med Coll, Sch Pharm, Wuhan, Peoples R China
Peking Univ First Hosp, Dept Dermatol, Beijing Key Lab Mol Diag Dermatoses, Beijing, Peoples R China
Natl Clin Res Ctr Skin & Immune Dis, Beijing, Peoples R China
Capital Med Univ, Beijing Childrens Hosp, Natl Ctr Childrens Hlth, Dept Dermatol, Beijing, Peoples R China
Zhengzhou Univ, Affiliated Childrens Hosp, Henan Childrens Hosp, Zhengzhou Childrens Hosp,Dept Dermatol, Zhengzhou, Peoples R China
KeywordsPHOSPHATE BACKBONE
RARE MUTATIONS
DNA
PCR
OLIGONUCLEOTIDES
ENRICHMENT
PHOSPHOROTHIOATION
ENDONUCLEASE
GENERATION
NUCLEASES
Issue Date2023
PublisherNATURE BIOMEDICAL ENGINEERING
AbstractDNA mutations occurring at low frequencies can be more easily detected by first excising wild-type DNA strands via the endonuclease deoxyribonuclease I, guided by single-stranded phosphorothioated DNA. The efficiency of DNA-enrichment techniques is often insufficient to detect mutations that occur at low frequencies. Here we report a DNA-excision method for the detection of low-frequency mutations in genomic DNA and in circulating cell-free DNA at single-nucleotide resolution. The method is based on a competitive DNA-binding-and-digestion mechanism, effected by deoxyribonuclease I (DNase) guided by single-stranded phosphorothioated DNA (sgDNase), for the removal of wild-type DNA strands. The sgDNase can be designed against any wild-type DNA sequences, allowing for the uniform enrichment of all the mutations within the target-binding region of single-stranded phosphorothioated DNA at mild-temperature conditions. Pretreatment with sgDNase enriches all mutant strands with initial frequencies down to 0.01% and leads to high discrimination factors for all types of single-nucleotide mismatch in multiple sequence contexts, as we show for the identification of low-abundance mutations in samples of blood or tissue from patients with cancer. The method can be coupled with next-generation sequencing, droplet digital polymerase chain reaction, Sanger sequencing, fluorescent-probe-based assays and other mutation-detection methods.
URIhttp://hdl.handle.net/20.500.11897/689357
ISSN2157-846X
DOI10.1038/s41551-023-01072-8
IndexedSCI(E)
Appears in Collections:化学与分子工程学院
生物有机与分子工程教育部重点实验室
第一医院

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