Target DNA detection and quantitation on a single cell with single base resolution

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• Accurate DNA detection at the single-cell level is crucial for understanding cellular heterogeneity and disease mechanisms.
• Existing methods often lack the sensitivity or resolution required for precise genetic analysis in individual cells.

Purpose of the Study:

• To develop and validate a novel method for ultra-sensitive DNA detection in single cells with single base resolution.
• To demonstrate the capability of the method for identifying specific DNA sequences, including oncoviral inserts and genetic variations.

Main Methods:

• Integration of peptide nucleic acid (PNA) openers for targeted DNA tagging.
• Utilizing isothermal rolling circle amplification (RCA) for signal amplification.
• Implementing a cells-in-flow format with fluorescence-based detection and flow cytometry.

Main Results:

• Achieved single base resolution for DNA detection in single cells.
• Demonstrated sensitive detection of oncoviral DNA inserts in cancer cell lines.
• Successfully performed quantitative detection of single-nucleotide polymorphisms (SNPs) and copy-number variations (CNVs) under isothermal conditions.

Conclusions:

• The developed method offers a powerful tool for ultra-sensitive DNA characterization at the single-cell level.
• Its ability to provide single base resolution and detect genetic variations opens new avenues for diagnostics and research.
• The cells-in-flow format and isothermal amplification make it suitable for various biological applications requiring precise genetic analysis.