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Related Experiment Videos

High-density multiplex detection of nucleic acid sequences: oligonucleotide ligation assay and sequence-coded

P D Grossman1, W Bloch, E Brinson

  • 1Applied Biosystems Division, Perkin Elmer Corporation, Foster City, CA 94404.

Nucleic Acids Research
|October 25, 1994
PubMed
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This study introduces a semi-automated method for analyzing multiple nucleic acid sequences, like the cystic fibrosis transmembrane regulator (CFTR) gene. It enables efficient large-scale genetic analysis with unique identifiers for each target.

Area of Science:

  • Molecular Biology
  • Genetics
  • Biotechnology

Background:

  • Multiplex analysis of nucleic acid sequences is crucial for genetic research.
  • Existing methods may face limitations in scale and automation.
  • Accurate allelic discrimination in polymorphic genes like CFTR is essential.

Purpose of the Study:

  • To develop a non-isotopic, semi-automated method for large-scale multiplex nucleic acid sequence analysis.
  • To enable efficient allelic discrimination in highly polymorphic genes.
  • To utilize oligonucleotide ligation assay (OLA) with novel mobility modifiers.

Main Methods:

  • A multiplex oligonucleotide ligation assay (OLA) was employed.
  • Fluorescently tagged probes and oligomeric non-nucleotide mobility modifiers were used.

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  • Products were resolved via electrophoresis under denaturing conditions with fluorescence detection.
  • Mobility modifiers were synthesized using automated phosphoramidite chemistry.

    • Main Results:

    • Each OLA product exhibited unique electrophoretic mobility determined by ligated oligonucleotides and assigned mobility modifiers.
    • Mobility modifiers provided a wider range than unmodified oligonucleotides.
    • Mobility modifiers minimally impacted probe-target duplex Tm and annealing kinetics.
    • Negligible effects on OLA yield and specificity were observed.

    Conclusions:

    • The developed semi-automated OLA method is effective for large-scale multiplex nucleic acid analysis.
    • This technique is particularly valuable for allelic discrimination in polymorphic genes such as CFTR.
    • The use of mobility modifiers enhances multiplexing capabilities without compromising assay performance.