Improved sensitivity for solid-support invasive cleavage reactions with flow cytometry analysis.

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• Accurate detection of nucleic acid variations is crucial for genetic analysis.
• Existing methods may lack the sensitivity or throughput for certain applications.
• Single nucleotide polymorphisms (SNPs) require highly specific detection methods.

Purpose of the Study:

• To develop a high-sensitivity assay for discriminating nucleic acid targets with single nucleotide differences.
• To establish a solid-support invasive cleavage reaction in a small reaction-volume format.
• To evaluate the assay's performance for genomic DNA analysis, including SNPs.

Main Methods:

• Utilized a solid-support invasive cleavage reaction with tethered oligonucleotides forming a tripartite substrate.
• Employed Cleavase, a structure-specific 5'-nuclease, for target cleavage and signal generation.
• Analyzed reactions using fluorometry for real-time data and flow cytometry for endpoint detection on microspheres.

Main Results:

• Achieved high-sensitivity discrimination of single nucleotide differences at target concentrations as low as 2 amol/assay.
• Demonstrated clear distinction of single base mutations.
• Showcased the assay's capability to detect target concentrations below 200 fM in a 10 microL reaction volume using 3000 microspheres.

Conclusions:

• The solid-support invasive cleavage reaction offers a sensitive and specific method for nucleic acid target discrimination.
• The microsphere-based assay, particularly with flow cytometry, is suitable for high-throughput genomic applications like SNP analysis.
• This assay configuration provides a valuable tool for molecular diagnostics and genetic research.