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

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.
• 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.