A simple and ultrasensitive fluorescence assay for single-nucleotide polymorphism.

Area of Science:

• Molecular Biology
• Biochemistry
• Analytical Chemistry

Background:

• Single-nucleotide polymorphism (SNP) detection is crucial for genetic analysis and diagnostics.
• Existing SNP detection methods often require complex procedures, labels, or lack sufficient sensitivity and selectivity.
• Development of simple, efficient, and ultrasensitive nucleic acid detection techniques is highly desirable.

Purpose of the Study:

• To develop a simple, label-free, and highly efficient nucleic acid amplification technique for ultrasensitive SNP detection.
• To establish a fluorescence-based detection method for pyrophosphate molecules generated during amplification.
• To demonstrate the assay's capability for detecting low concentrations of mutant alleles within wild-type samples.

Main Methods:

• Design and circularization of a padlock probe that hybridizes specifically to the target mutant gene.
• Initiation of branched rolling circle amplification (BRCA) using the circularized probe, generating extensive DNA strands and pyrophosphate.
• Sensitive fluorescence detection of pyrophosphate using a terpyridine-Zn(II) complex, correlating fluorescence intensity with mutant gene concentration.

Main Results:

• The assay achieved an ultrasensitive detection limit of 0.1 pM for SNP.
• An excellent selectivity factor of 1000 was obtained, distinguishing between mutant and wild-type sequences.
• As little as 0.1% mutant DNA within a wild-type background was successfully detected.

Conclusions:

• The developed method offers a simple, label-free, and highly sensitive approach for SNP detection.
• The combination of BRCA and pyrophosphate fluorescence detection provides a robust platform for genetic variation analysis.
• This technique presents a potentially viable alternative for routine SNP analysis in various applications.