Fluorescence detection of single nucleotide polymorphisms using a universal molecular beacon.

Area of Science:

• Biotechnology
• Molecular Biology
• Analytical Chemistry

Background:

• Single nucleotide polymorphisms (SNPs) are crucial genetic markers.
• Existing methods for SNP detection can be complex and lack selectivity.
• Molecular beacons (MBs) offer a platform for nucleic acid detection.

Purpose of the Study:

• To develop a novel, simple, and selective assay for SNP detection.
• To utilize mercury(II) ions (Hg(2+)) and a universal molecular beacon for SNP detection.
• To investigate the mechanism of Hg(2+)-DNA complex formation and its effect on MB conformation and fluorescence.

Main Methods:

• Design and synthesis of a T(7)-MB probe with FAM and DABCYL fluorophores.
• Utilizing Hg(2+)-T-Hg(2+)-T bonding to induce MB conformational changes.
• Measuring changes in fluorescence resonance energy transfer (FRET) efficiency.
• Optimizing assay conditions including MB concentration, salt concentration, Hg(2+) concentration, and buffer pH.
• Evaluating the assay's linearity, limit of detection, and selectivity for perfectly matched DNA.

Main Results:

• Hg(2+) binding to the T(7)-MB probe induced a conformational change, decreasing fluorescence intensity.
• The assay demonstrated a linear response between fluorescence intensity and perfectly matched DNA concentration from 2-30 nM (R(2) = 0.991).
• A low limit of detection (0.5 nM) was achieved at optimal conditions.
• The developed probe exhibited higher selectivity for DNA compared to conventional MBs.

Conclusions:

• A simple and novel assay for SNP detection using a universal MB and Hg(2+) was successfully developed.
• The assay leverages Hg(2+)-induced conformational changes in the MB for sensitive and selective DNA detection.
• This approach offers a promising alternative for SNP genotyping and other nucleic acid detection applications.