Gold nanoparticle-based electrophoresis-free colorimetric detection method for allele-specific PCR-SNP genotyping

Area of Science:

• Molecular Biology
• Nanotechnology
• Genetics

Background:

• Conventional allele-specific PCR (AS-PCR) genotyping relies on gel electrophoresis and ethidium bromide (EtBr), which are expensive and pose health risks.
• There is a need for cost-effective, safer, and simpler genotyping methods, especially in resource-limited settings.
• Ethidium bromide is a toxic dye requiring specialized equipment and posing health hazards.

Purpose of the Study:

• To develop a simple, cost-effective, and safe colorimetric genotyping method using gold nanoparticles (AuNPs).
• To replace traditional gel electrophoresis and ethidium bromide (EtBr) for AS-PCR genotyping.
• To enable visual detection of amplified and non-amplified PCR products.

Main Methods:

• Developed a colorimetric genotyping assay using gold nanoparticles (AuNPs) and unmodified primers.
• Utilized a PCR mastermix without a dye, observing color changes based on amplicon presence.
• Verified results using Transmission Electron Microscopy (TEM) and compared with agarose gel electrophoresis.

Main Results:

• Amplified PCR products appeared red, while non-amplified products appeared blue, due to AuNPs sequestration or aggregation.
• The method successfully genotyped six single-nucleotide polymorphisms (SNPs) in buffaloes, matching agarose gel electrophoresis results.
• 15μl of AuNPs solution was sufficient for detecting an amplicon in 5μl of PCR product.

Conclusions:

• The AS-PCR combined with AuNPs offers an easy visual detection method for amplified and non-amplified PCR products.
• This AuNPs-based method is a cost-effective and safer alternative to traditional gel electrophoresis and EtBr.
• The presented method has the potential to replace agarose gel electrophoresis, EtBr, and UV-transilluminators in genotyping.