Cost-Efficient Sequence-Based Nonextensible Oligonucleotide in Real-Time PCR and High-Throughput Sequencing.

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• Accurate detection of low-abundance disease mutations is crucial for research and clinical diagnostics.
• Current methods often rely on chemically modified blocking oligonucleotides, which are expensive and difficult to synthesize.
• There is a need for cost-effective and efficient blocking strategies for sensitive mutation detection.

Purpose of the Study:

• To introduce a novel, chemistry-free blocking oligonucleotide design, termed nonextensible oligonucleotide (NEO).
• To evaluate the efficacy of NEOs in preventing polymerase extension and enhancing mutation detection.
• To demonstrate the utility of NEOs in various quantitative PCR and next-generation sequencing applications.

Main Methods:

• NEOs were designed with secondary structures at the 3' end to create steric hindrance, blocking enzymatic extension.
• The performance of NEOs was tested with Taq and high-fidelity DNA polymerases.
• NEOs were integrated into blocker displacement amplification (BDA) qPCR, multiplex BDA (mBDA) for NGS, and quantitative BDA (QBDA) for NGS.

Main Results:

• NEOs effectively inhibited DNA polymerase extension, comparable to chemical modifications.
• QBDA using NEO blockers showed 100% concordance with droplet digital PCR (ddPCR) for clinical FFPE samples.
• NEOs demonstrated utility in optimizing plex uniformity for an 80-plex NGS panel by modulating PCR amplification efficiency.

Conclusions:

• NEOs provide an effective, chemistry-free alternative for blocking unwanted DNA amplification in molecular assays.
• This technology offers a cost-effective and efficient solution for sensitive detection of rare mutations.
• NEOs show broad applicability in various molecular diagnostic and research platforms, including NGS and qPCR.