Hairpin Structure Facilitates Multiplex High-Fidelity DNA Amplification in Real-Time Polymerase Chain Reaction.

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• Detecting rare DNA variants is crucial for early cancer diagnosis and identifying drug resistance mutations.
• Standard quantitative polymerase chain reaction (qPCR) methods often lack sensitivity due to high DNA polymerase misincorporation rates.
• High-fidelity (HiFi) DNA polymerases offer superior accuracy but lack suitable probe designs for multiplexed qPCR variant detection.

Purpose of the Study:

• To develop a novel probe-based fluorescence indicator system for multiplexed HiFi qPCR.
• To enable the application of HiFi DNA polymerases in sensitive DNA variant detection.
• To improve the limit of detection (LoD) for rare DNA variants.

Main Methods:

• Designed an occlusion system comprising a 5'-overhang primer with fluorophore and a hairpin probe with a 3' quencher.
• Integrated the occlusion system into HiFi qPCR reactions.
• Combined the occlusion system with a variant-enrichment method.

Main Results:

• The occlusion system enabled multiplexed HiFi qPCR reactions.
• The system demonstrated compatibility with existing variant-enrichment techniques.
• A significant improvement in the limit of detection (LoD), up to 10-fold, was achieved.
• The occlusion system effectively served as a fluorescence indicator for HiFi qPCR.

Conclusions:

• The occlusion system is an efficient fluorescence indicator for HiFi qPCR.
• This system facilitates the use of HiFi DNA polymerases for enhanced DNA variant detection sensitivity.
• The improved sensitivity aids in early cancer diagnosis and drug resistance mutation identification.