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Related Concept Videos

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Detection of Rare Genomic Variants from Pooled Sequencing Using SPLINTER
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Analysis of single nucleotide polymorphisms with solid phase invasive cleavage reactions.

P Wilkins Stevens1, J G Hall, V Lyamichev

  • 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

Nucleic Acids Research
|August 16, 2001
PubMed
Summary

This study demonstrates a solid-phase invasive cleavage assay using microparticles for genomic applications. This method effectively distinguishes single nucleotide polymorphisms (SNPs) with high sensitivity.

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Area of Science:

  • Biotechnology
  • Molecular Biology
  • Genomics

Background:

  • The solution phase invasive cleavage assay is a signal amplification method for distinguishing nucleic acids with single base mutations.
  • Adaptation to a solid phase platform is crucial for microarray and multiplex applications.

Purpose of the Study:

  • To demonstrate the feasibility of performing the invasive cleavage reaction on a solid phase using microparticles.
  • To adapt the invasive cleavage assay for solid-phase platforms like microarrays.

Main Methods:

  • Utilized microparticles as the capture surface and fluorescence resonance energy transfer (FRET) for detection.
  • Configured overlapping oligonucleotides (probe and upstream) on target nucleic acids for cleavage by a 5'-nuclease.
  • Investigated different configurations of oligonucleotide attachment to microparticles and the use of long tethers.

Main Results:

  • Effective cleavage of probe oligonucleotides was achieved with particle-bound reagents and when both oligonucleotides were attached to the solid phase.
  • Positioning probe oligonucleotides away from the particle surface using long tethers enhanced signal and reaction rates.
  • The particle-based assay distinguished ApoE Cys158 and Arg158 alleles at concentrations as low as 100 amol/assay (0.5 pM).

Conclusions:

  • The solid-phase invasive cleavage reaction using microparticles is feasible and effective for genomic applications.
  • This method offers a robust platform for sensitive detection of single nucleotide polymorphisms (SNPs).
  • The assay demonstrates potential for multiplex applications requiring high specificity and sensitivity.