Nanopore Identification of Single Nucleotide Mutations in Circulating Tumor DNA by Multiplexed Ligation.
Clin Chem
; 67(5): 753-762, 2021 04 29.
Article
em En
| MEDLINE
| ID: mdl-33496315
ABSTRACT
BACKGROUND:
Circulating tumor DNAs (ctDNAs) are highly promising cancer biomarkers, potentially applicable for noninvasive liquid biopsy and disease monitoring. However, to date, sequencing of ctDNAs has proven to be challenging primarily due to small sample size and high background of fragmented cell-free DNAs (cfDNAs) derived from normal cells in the circulation, specifically in early stage cancer.METHODS:
Solid-state nanopores (ssNPs) have recently emerged as a highly efficient tool for single-DNA sensing and analysis. Herein, we present a rapid nanopore genotyping strategy to enable an amplification-free identification and classification of ctDNA mutations. A biochemical ligation detection assay was used for the creation of specific fluorescently-labelled short DNA reporter molecules. Color conjugation with multiple fluorophores enabled a unique multi-color signature for different mutations, offering multiplexing potency. Single-molecule readout of the fluorescent labels was carried out by electro-optical sensing via solid-state nanopores drilled in titanium oxide membranes.RESULTS:
As proof of concept, we utilized our method to detect the presence of low-quantity ERBB2 F310S and PIK3Ca H1047R breast cancer mutations from both plasmids and xenograft mice blood samples. We demonstrated an ability to distinguish between a wild type and a mutated sample, and between the different mutations in the same sample.CONCLUSIONS:
Our method can potentially enable rapid and low cost ctDNA analysis that completely circumvents PCR amplification and library preparation. This approach will thus meet a currently unmet demand in terms of sensitivity, multiplexing and cost, opening new avenues for early diagnosis of cancer.Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Neoplasias da Mama
/
Nanoporos
/
DNA Tumoral Circulante
Tipo de estudo:
Diagnostic_studies
/
Prognostic_studies
/
Screening_studies
Limite:
Animals
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Female
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Humans
Idioma:
En
Ano de publicação:
2021
Tipo de documento:
Article