Quantitative and discriminative analysis of nucleic acid samples using luminometric nonspecific nanoparticle methods

Area of Science:

• Biochemistry
• Analytical Chemistry
• Molecular Biology

Background:

• Quantifying nucleic acids is crucial in molecular biology.
• Existing methods often require sequence information or lack sensitivity for different DNA forms.
• Developing sequence-independent nucleic acid quantification assays is highly desirable.

Purpose of the Study:

• To develop homogeneous assays for quantifying nucleic acids using luminescence quenching and time-resolved luminescence resonance energy transfer (TR-LRET).
• To achieve sequence-independent detection of both single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA).
• To establish a multiplexed assay for simultaneous quantification of total DNA and the ratio of ssDNA to dsDNA.

Main Methods:

• Utilized europium nanoparticles and a soluble quencher for luminescence quenching assay.
• Employed time-resolved luminescence resonance energy transfer (TR-LRET) from europium nanoparticles to an acceptor dye.
• Developed a single-tube assay combining TR-LRET with a dsDNA-selective dye.

Main Results:

• Achieved equal sensitivity for ssDNA and dsDNA, unlike fluorescent dye-binding methods.
• Established a detection limit of 60 pg for the luminescence quenching assay.
• Demonstrated high precision with average coefficients of variation of 5% (quenching) and 8% (TR-LRET).
• Successfully developed a multiplexed assay for DNA and ssDNA/dsDNA ratio determination.

Conclusions:

• Developed novel, sequence-independent homogeneous assays for nucleic acid quantification.
• The developed assays offer sensitive and precise detection of ssDNA and dsDNA.
• The multiplexed TR-LRET assay provides a unique capability for analyzing DNA composition in a single tube, surpassing current commercial offerings.