Multiplexed DNA detection with a composite molecular beacon based on guanine-quenching

Area of Science:

• Molecular Biology
• Biochemistry
• Analytical Chemistry

Background:

• Accurate detection of tumor-suppressor genes like p16 and p53 is crucial for cancer diagnostics.
• Existing multiplexed DNA detection methods can be complex and time-consuming.

Purpose of the Study:

• To develop a novel, efficient, and cost-effective multiplexed DNA detection method.
• To enable simultaneous detection of two distinct DNA targets using a single assay.

Main Methods:

• A composite molecular beacon (MB) probe design incorporating guanine-quenching was developed.
• Synchronous fluorescence analysis was employed for signal detection.
• The method was validated using exon segments of p16 (T1) and p53 (T2) tumor-suppressor genes.

Main Results:

• The composite MB probe demonstrated simultaneous fluorescence recovery of FAM and TAMRA upon target DNA binding.
• Linear relationships were observed between fluorescence intensity and target DNA concentration (5 × 10⁻¹¹ to 5.5 × 10⁻⁹ M).
• Low detection limits were achieved: 4 × 10⁻¹¹ M for T1 and 3 × 10⁻¹¹ M for T2.

Conclusions:

• The developed guanine-quenching composite MB method allows for sensitive and simultaneous detection of multiple DNA targets.
• This approach offers advantages in terms of probe synthesis, cost, and analytical time compared to existing methods.
• The method shows potential for application in real sample analysis and detection of arbitrary DNA sequences.