Colorimetric detection of single-nucleotide mutations based on rolling circle amplification and G-quadruplex-based DNAzyme.
Anal Methods
; 16(34): 5785-5792, 2024 Aug 29.
Article
in En
| MEDLINE
| ID: mdl-39140250
ABSTRACT
In this work, we proposed a sensitive and selective colorimetric assay for single nucleotide mutation (SNM) detection combining rolling circle amplification (RCA) and G-quadruplex/hemin DNAzyme complex formation. In the detection principle, the first step involves ssDNA hybridization with a padlock probe (PLP) DNA, which can discriminate a single base mismatch. The successful ligation is followed by an RCA event to generate an abundance of G-quadruplexes (GQ-RCA) which are then transformed into a DNAzyme (G-quadruplex/hemin complex) by the addition of hemin. The color change from colorless 3,3',5,5'-tetramethylbenzidine (TMB) into colored oxTMB when hydrogen peroxide (H2O2) is added indicated the presence of a mutation. The assay had a limit of detection (LOD) of 2.14 pM. Mutations in samples from breast cancer patients were successfully detected with an accuracy of 100% when compared to Sanger sequencing results. The method is easily applicable even in resource poor setting regions given that it doesn't require any sophisticated or expensive instruments, and the signal readout is detectable simply by the naked eye. Our assay might be a useful tool for genetic analysis and clinical molecular diagnosis for breast cancer risk assessment and early detection.
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Colorimetry
/
DNA, Catalytic
/
Nucleic Acid Amplification Techniques
/
G-Quadruplexes
Limits:
Female
/
Humans
Language:
En
Journal:
Anal Methods
Year:
2024
Document type:
Article
Affiliation country:
China
Country of publication:
United kingdom