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Nucleic Acids Res DOI:10.1093/nar/gkz870

A nuclease-polymerase chain reaction enables amplification of probes used for capture-based DNA target enrichment.

Publication TypeJournal Article
Year of Publication2019
AuthorsLeong, KWai, Yu, F, Adalsteinsson, VA, Reed, S, Gydush, G, Ladas, I, Li, J, Tantisira, KG, Makrigiorgos, GMike
JournalNucleic Acids Res
Date Published2019 Oct 10
ISSN1362-4962
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.

DOI10.1093/nar/gkz870
Pubmed

http://www.ncbi.nlm.nih.gov/pubmed/31598677?dopt=Abstract

Alternate JournalNucleic Acids Res.
PubMed ID31598677