An optimized method for PCR-based genotyping to detect human APOE polymorphisms
Leila Najd-Hassan-Bonab1, Mehdi Hedayati1, Seyed Abolhassan Shahzadeh Fazeli2
1Cellular and Molecular Endocrine Research Center, Research Institute for Endocrine Sciences, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
Heliyon
|November 13, 2023
Summary
We optimized a Tetra-ARMS PCR method for accurate Apolipoprotein E (APOE) genotyping. This simple, rapid, and sensitive assay is suitable for large-scale studies and low-resource labs.
Area of Science:
- Genetics
- Molecular Biology
- Biochemistry
Background:
- Apolipoprotein E (APOE) gene polymorphisms are linked to various diseases, including neurodegenerative conditions and type 2 diabetes.
- Accurate APOE genotyping is crucial for assessing individual disease risk.
- The APOE gene has two key single nucleotide polymorphisms (SNPs) at rs429358 and rs7412.
Purpose of the Study:
- To optimize the tetra amplification refractory mutation system (Tetra-ARMS) PCR method for efficient APOE genotyping.
- To develop a sensitive and accurate assay for detecting APOE mutations.
Main Methods:
- Optimized Tetra-ARMS PCR conditions, including cycle parameters and HiFidelity enzyme concentration.
- Eliminated the need for hazardous reagents like dimethyl sulfoxide (DMSO) in the PCR protocol.
- Validated the assay by comparing results with established RFLP and chip-typing methods.
Main Results:
- The optimized Tetra-ARMS PCR achieved 100% accuracy and sensitivity.
- The method successfully amplified DNA fragments in just 25 cycles without DMSO.
- Achieved 100% consistency with chip-typing and 91% consistency with RFLP.
Conclusions:
- The developed Tetra-ARMS PCR method provides a simple, accurate, and rapid approach for APOE genotyping.
- This assay is suitable for analyzing large sample sizes efficiently.
- The method is adaptable for use in laboratories with limited technological resources.
