Genotyping single-nucleotide polymorphisms by the invader assay with dual-color fluorescence polarization detection

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• The Polymerase Chain Reaction (PCR)-Invader assay is a sensitive and specific method for genotyping single-nucleotide polymorphism (SNP) markers.
• Existing methods can be streamlined and made more cost-effective for broader application.

Purpose of the Study:

• To improve the PCR-Invader assay by enabling simultaneous genotyping of both alleles for each SNP in a single reaction.
• To reduce the cost of the PCR-Invader assay by implementing fluorescence polarization (FP) as the detection method.

Main Methods:

• Developed a streamlined PCR-Invader protocol to assay both alleles for each SNP concurrently.
• Integrated fluorescence polarization (FP) detection using fluorescein and TAMRA-labeled signal probes.
• Incubated PCR products with Invader oligonucleotide, primary probes, signal probes, and Cleavase VIII enzyme, followed by FP measurements.

Main Results:

• Successfully genotyped 88 individuals across 10 SNPs, generating 880 genotypes with a low initial 'no call' rate of 3.2%.
• All samples failing initial genotyping produced clear results upon re-amplification.
• Achieved 100% concordance between the improved PCR-Invader assay and the template-directed dye-terminator incorporation assay with FP.

Conclusions:

• The modifications simplify the PCR-Invader assay, making it more user-friendly.
• The adoption of FP detection significantly reduces the cost of the assay.
• These enhancements make the PCR-Invader assay a more practical and cost-effective solution for high-throughput SNP genotyping.