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• W2980245990 abstract "DNA target enrichment via hybridization capture is a commonly adopted approach which remains expensive due in-part to using biotinylated-probe panels. Here we provide a novel isothermal amplification reaction to amplify rapidly existing probe panels without knowledge of the sequences involved, thereby decreasing a major portion of the overall sample preparation cost. The reaction employs two thermostable enzymes, BST-polymerase and duplex-specific nuclease DSN. DSN initiates random 'nicks' on double-stranded-DNA which enable BST to polymerize DNA by displacing the nicked-strand. Displaced strands re-hybridize and the process leads to an exponential chain-reaction generating biotinylated DNA fragments within minutes. When starting from single-stranded-DNA, DNA is first converted to double-stranded-DNA via terminal-deoxynucleotidyl-transferase (TdT) prior to initiation of BST-DSN reaction. Biotinylated probes generated by TdT-BST-DSN (TBD) reactions using panels of 33, 190 or 7186 DNA targets are used for hybrid-capture-based target enrichment from amplified circulating-DNA, followed by targeted re-sequencing. Polymerase-nuclease isothermal-chain-reactions generate random amplified probes with no apparent sequence dependence. One round of target-capture using TBD probes generates a modest on-target sequencing ratio, while two successive rounds of capture generate >80% on-target reads with good sequencing uniformity. TBD-reactions generate enough capture-probes to increase by approximately two to three orders-of-magnitude the target-enrichment experiments possible from an initial set of probes." @default.
• W2980245990 created "2019-10-18" @default.
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• W2980245990 date "2019-10-10" @default.
• W2980245990 modified "2023-10-15" @default.
• W2980245990 title "A nuclease-polymerase chain reaction enables amplification of probes used for capture-based DNA target enrichment" @default.
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• W2980245990 doi "https://doi.org/10.1093/nar/gkz870" @default.
• W2980245990 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/6902007" @default.
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