Detection of low-frequency mutations in clinical samples by increasing mutation abundance via the excision of wild-type sequences.
Nat Biomed Eng
; 7(12): 1602-1613, 2023 Dec.
Article
em En
| MEDLINE
| ID: mdl-37500748
ABSTRACT
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.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Neoplasias
Tipo de estudo:
Diagnostic_studies
/
Prognostic_studies
Limite:
Humans
Idioma:
En
Revista:
Nat Biomed Eng
Ano de publicação:
2023
Tipo de documento:
Article