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Related Experiment Videos

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

P Wilkins Stevens1, K V N Rao, J G Hall

  • 1Department of Biomedical Engineering, Robert R. McCormick School of Engineering and Applied Science, Northwestern University, 2145 Sheridan Road, Evanston, IL 60208-3107, USA. pwilkins@northwestern.edu

Biotechniques
|January 28, 2003
PubMed
Summary
This summary is machine-generated.

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This study introduces a highly sensitive solid-support invasive cleavage reaction for detecting single nucleotide differences in nucleic acids. The assay achieves high sensitivity at low target concentrations, enabling precise genetic 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.

Related Experiment Videos

  • 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.