One pot PCR genotyping of ApoE for diagnosing Alzheimer’s disease
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Apolipoprotein E (apoE) genotyping is a valuable tool for assessing genetic risk associated with Alzheimer’s disease (AD). The ε4 allele is strongly linked to an increased risk and earlier onset of AD symptoms. Individuals carrying one (heterozygous) or two (homozygous) copies of the ε4 allele are at significantly higher risk compared to those without it. In contrast, the ε2 allele may offer a protective effect, reducing the likelihood of developing the disease. Many studies have reported the use of PCR-based methods for apoE genotyping. However, current approaches typically require at least two tubes—and often more—to distinguish among the six possible genotypes using mixtures of allele-specific primers. Consolidating the assay into a single tube can substantially reduce assay time, minimize manual labor, and increase throughput. To address this need, we developed a one-pot PCR assay platform for apoE genotyping, utilizing an RNA-DNA hybrid CataCleave probe. We provide a detailed explanation of the assay’s principles, including its limit of detection, specificity, resistance to interference from other serum components, and precision. Collectively, this information enables other researchers to adopt our streamlined, single-tube apoE genotyping method with ease.
