Simultaneous multiple single nucleotide polymorphism detection based on click chemistry combined with DNA-encoded...

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• Single nucleotide polymorphisms (SNPs) are vital biomarkers for disease diagnosis, prognosis, and pathogenesis.
• Detecting multiple SNPs reliably is essential due to the association of various SNPs with specific diseases.

Purpose of the Study:

• To develop a sensitive and reliable method for detecting single nucleotide polymorphisms (SNPs).
• To establish an enzyme-free approach for multiplexed SNP detection in a single reaction.

Main Methods:

• Utilized an enzyme-free, DNA template-directed click reaction, specifically Cu(i)-catalyzed alkyne-azide cycloaddition (CuAAC).
• Oligonucleotide probes were labeled with 5'-alkyne and 3'-azide groups, forming a triazole backbone mimicking a phosphodiester linkage upon target DNA binding.
• Detection was performed using capillary gel electrophoresis with laser-induced fluorescence (CGE-LIF).

Main Results:

• Achieved sensitive detection of target DNA down to 25 fmol in 50 μL.
• Demonstrated that SNPs significantly influence click reaction efficiency, enabling detection of as little as 0.5% SNP.
• Successfully performed multiplexed SNP detection in a single tube by encoding different probe lengths for distinct SNP sites.

Conclusions:

• The developed enzyme-free click reaction offers a sensitive and specific method for SNP detection.
• This approach facilitates multiplexed SNP analysis, holding promise for advancing disease biomarker discovery and diagnostics.