One pot PCR genotyping of ApoE for diagnosing Alzheimer’s disease

One pot PCR genotyping of ApoE for diagnosing Alzheimer’s disease | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article One pot PCR genotyping of ApoE for diagnosing Alzheimer’s disease Hyemin Kim, June Hahk Bae, Mihwa Yang, Younjoo Yang, Woolim Cha, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6415246/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract 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. Biological sciences/Biological techniques/Genetic techniques/Pcr based techniques Health sciences/Diseases/Neurological disorders/Dementia/Alzheimers disease ApoE genotype Real-time PCR Alzheimer’s disease CataCleave probe Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Apolipoprotein E (apoE) genotyping is a critical tool for assessing genetic risk associated with Alzheimer’s disease (AD), particularly late-onset Alzheimer’s, the most common form of the condition 1 . The APOE gene has three major alleles—ε2, ε3, and ε4—and each individual inherits one allele from each parent. These alleles influence the likelihood of developing AD. Notably, the ε4 allele is strongly associated with an increased risk and earlier onset of AD symptoms. Individuals carrying one copy (heterozygous) or two copies (homozygous) of ε4 face a significantly higher risk compared to those without the allele 2 . In contrast, the ε2 allele may offer a protective effect, lowering the risk of disease development. ApoE genotyping is especially valuable for identifying individuals who may benefit from early monitoring or participation in clinical trials targeting high-risk populations 3 . It also enhances our understanding of the biological mechanisms underlying AD, as apoE plays a role in amyloid-beta metabolism, lipid transport, and neuronal repair 4 . While apoE genotyping is not a standalone diagnostic tool, it becomes particularly informative when combined with clinical evaluations, neuroimaging, and biomarker analyses. Together, these tools can improve risk assessment and support personalized prevention or intervention strategies 5 . Current PCR-based methods for apoE genotyping typically require at least two reaction tubes—and often more—to distinguish among the six possible genotypes using combinations of allele-specific primers 6 . Consolidating the assay into a single-tube format would greatly reduce assay time, minimize manual labor, and improve throughput. To address this, we developed a one-pot PCR assay platform for apoE genotyping utilizing an RNA-DNA hybrid CataCleave probe. This probe is a chimeric DNA-RNA-DNA structure incorporating two internal fluorophores 7 . Upon hybridization to the target DNA, ribonuclease H (RNase H) cleaves the RNA portion of the probe, causing the resulting fragments to dissociate. This cleavage restores the donor fluorescence signal, allowing for precise and efficient detection of target sequences. Results Assay Principle The APOE gene, located on chromosome 19q13.2, encodes a 299-amino acid apolipoprotein E protein, which exists in three major isoforms—ApoE2, ApoE3, and ApoE4—distinguished by two amino acid substitutions at positions 112 and 158 8 . The codons TGC and CGC encode cysteine and arginine, respectively. Consequently, the isoforms differ by having either Cys or Arg at these positions (Fig. 1). To discriminate among the six possible apoE genotypes, we developed a panel of four CataCleave probes, each labeled with a unique 5′ fluorescent reporter dye and a common 3′ quencher. The probes were designated as follows: 112T (FAM), 112C (HEX), 158T (Texas Red), and 158C (Cy5). A single pair of common forward and reverse primers was designed to amplify the region encompassing these polymorphisms (Fig. 2). Each probe contains a single ribonucleotide (either rU or rC), positioned to hybridize with a complementary base (A or G) in the target DNA (Table 1). Upon perfect base pairing, the RNA-DNA hybrid is cleaved by RNase H2, resulting in probe fragmentation and fluorescence signal restoration. This enables real-time detection of allele-specific amplification. To validate the assay, genomic DNA was extracted from cell lines with known apoE genotypes. Homozygous genotypes—ε2/ε2, ε3/ε3, and ε4/ε4—were confirmed using PC3, A549, and U937 cells, respectively. Heterozygous genotypes were reconstructed by mixing DNA from two cell lines (Table 2, Fig. 3). All homozygous genotypes were successfully amplified with Ct values <25. Heterozygous genotypes were detected with Ct values <38. The assay produced distinct fluorescence signatures for each genotype. For example, ε2/ε2 generated FAM and Texas Red signals, while ε2/ε4 produced FAM, Texas Red, HEX, and Cy5 signals. Limit of Detection, Interference, and Specificity The analytical sensitivity of the assay was evaluated by performing eight replicate reactions per genotype using serial dilutions of genomic DNA. The limit of detection (LoD) ranged from 0.05 to 0.5 ng/μL (Table 3). To assess potential interference, representative serum components—albumin, cholesterol, hemoglobin, and triglycerides—were added to the reactions. None of these interfered with assay performance (Table 4). Assay specificity was evaluated by spiking the reaction mixtures with nucleic acids from 11 bacterial and viral species. No cross-reactivity or false-positive results were observed, confirming high specificity (Table 5). Reproducibility was assessed by performing the assay in duplicate for seven consecutive days and twice daily over a 20-day period. In all cases, the coefficient of variation (CV) was <5% (data not shown), demonstrating excellent precision and inter-assay consistency. Clinical Sample Testing Following analytical validation, the assay was applied to 50 clinical samples collected from healthy volunteers. Genomic DNA was isolated from both peripheral blood and buccal swabs. As shown in Fig. 4 and Table 6, genotype calls were fully concordant across both sample types, demonstrating the assay’s robustness and suitability for clinical applications. Four distinct genotypes were reliably identified in the test population. Discussion In the present study, we developed a novel one-pot real-time quantitative PCR assay for apoE genotyping, utilizing our proprietary CataCleave probe technology. This platform, along with its enhanced version—Promer technology—enables sensitive and precise detection of single-point mutations, single nucleotide polymorphisms (SNPs), and microRNAs 9 , 10 . The core principle of both technologies lies in the specific cleavage of RNA-DNA hybrids by RNase H2, which occurs only upon perfect sequence complementarity. To discriminate among the six possible apoE genotypes, we designed four distinct CataCleave probes—each containing a strategically placed ribonucleotide—and a shared pair of forward and reverse primers. Depending on the genotype of the sample, the probes can produce up to four distinct fluorescent signals. For example, the ε2/ε4 genotype yields four separate fluorescence channels, each corresponding to a specific allele-probe match. Several commercial apoE genotyping kits are currently available; however, most employ two or more probe sets in separate reactions to independently assess the polymorphic positions at codons 112 and 158 of the APOE gene 11 . While some newer products offer one-pot PCR solutions, the assay principles and supporting validation data have not been disclosed. In contrast, our study presents comprehensive details regarding the assay mechanism, limit of detection, specificity, lack of interference from common serum components, and assay precision. We believe this transparency will enable other laboratories to readily adopt our approach and perform accurate apoE genotyping in a single-tube format. Despite the demonstrated reliability of our assay in clinical samples derived from both whole blood and buccal swabs, we did not observe any ε2/ε2 or ε4/ε4 homozygous genotypes in our sample set. This limitation likely reflects the relatively small sample size and the lower prevalence of these genotypes in the general population. Future studies involving larger and more diverse cohorts will be necessary to validate the assay's performance across all genotype categories and to better estimate the distribution of apoE alleles across different populations. Methods Reagents All DNA and RNA oligonucleotides were synthesized by Integrated DNA Technologies (Coralville, IA, USA). RNase H2 was purchased from BioAssay Co. Ltd (Daejeon, South Korea) or produced in-house. Briefly, cDNA of RNase H2 ( pyrococcus furiosus , GenBank: CP023154.1) was cloned into pET-28a plasmid and transformed into BL21(DE3)pLysS strain. Expression of the RNase H2 was induced with IPTG and purified using Ni-NTA affinity chromatography (ThermoFisher Scientific, Waltham, MA, USA) as previously reported 10 . Cell culture PC-3, A549 and U937 human cell lines were purchased from Korea Cell Line Bank ( http://cellbank.snu.ac.kr/main/ ). They were cultured in DMEM culture media containing 10% FBS and 1% antibiotics at 37 and 5% CO 2 according to instruction provided by KCLB. They were sub-cultured at 80% confluency using trypsin/EDTA at 1:8 ratio. Genomic DNA isolation Genomic DNA was extracted from 2ⅹ10 6 cells, whole blood or buccal swabs by using the DNA extraction kit (Accuprep®, Bioneer, Korea) according to the manufacturer’s instructions. DNA was diluted with nuclease-free water to 10 ng/µL for apoE genotyping analysis. Quantitative real-time PCR ApoE genotyping by Real Time PCR includes forward, reverse primer and four different kinds of CataCleave probes. Each PCR reaction mixture (15 µL) contained the following reagents: 1 × genotyping master mix, 0.5 µM of forward primer and 112T, 112C, 158T, 158C probes, 0.5 µM of reverse primer, the indicated amounts of the genomic DNA. The PCR amplification protocol was as follows: Initial activation of nTaq DNA polymerase at 95°C for 5 min, followed by 40 cycles with denaturation at 95°C for 15 sec, and annealing/extension at 65°C for 1 min. The fluorescence signals were collected during the annealing/extension step. FAM, HEX, TexasRed and Cy5 signals were read by using the CFX96 system (Bio-Rad Laboratories, Hercules, CA, USA). Clinical samples Whole blood and buccal swabs were collected from 50 healthy volunteers. Informed consent was obtained by Shihwa Medical Center, and the study was approved by the Institutional Review Board (Shihwa 2022 − 0124). All methods were performed in accordance with the relevant guidelines and regulations. After collecting blood sample, it was stored for up to 1 hour at room temperature (15 ~ 30℃) or up to 3 days at 2 ~ 10℃. For long-term storage, it was stored for up to one month if stored at -70℃ or below. For buccal swabs, it was stored in a transport medium for nucleic acid preservation for up to one month at 2 ~ 8 ℃. Statistical analysis Coefficient of variance (CV) were calculated using Excel software. Declarations Author contributions Kim H, Yang M, Yang Y, Cha W, Kwak T, Jeon S, Bae S, Son S, Lee E, and Yang H performed the experiments and collected the data. Bae JH and Nam YH designed the experiment and supervised the study. Shin JS analyzed the data and wrote the manuscript. Data availability statement The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request Competing interests Statement All authors declare no potential conflict of interest. Funding declaration This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. References Raulin, A. C. et al. ApoE in Alzheimer's disease: pathophysiology and therapeutic strategies. Mol Neurodegener 17 , 72, doi:10.1186/s13024-022-00574-4 (2022). Leoni, V. The effect of apolipoprotein E (ApoE) genotype on biomarkers of amyloidogenesis, tau pathology and neurodegeneration in Alzheimer's disease. Clin Chem Lab Med 49 , 375-383, doi:10.1515/CCLM.2011.088 (2011). Kim, J., Basak, J. M. & Holtzman, D. M. The role of apolipoprotein E in Alzheimer's disease. Neuron 63 , 287-303, doi:10.1016/j.neuron.2009.06.026 (2009). Huang, Y. & Mahley, R. W. Apolipoprotein E: structure and function in lipid metabolism, neurobiology, and Alzheimer's diseases. Neurobiol Dis 72 Pt A , 3-12, doi:10.1016/j.nbd.2014.08.025 (2014). Lumsden, A. L., Mulugeta, A., Zhou, A. & Hypponen, E. Apolipoprotein E (APOE) genotype-associated disease risks: a phenome-wide, registry-based, case-control study utilising the UK Biobank. EBioMedicine 59 , 102954, doi:10.1016/j.ebiom.2020.102954 (2020). Najd-Hassan-Bonab, L., Hedayati, M., Shahzadeh Fazeli, S. A. & Daneshpour, M. S. An optimized method for PCR-based genotyping to detect human APOE polymorphisms. Heliyon 9 , e21102, doi:10.1016/j.heliyon.2023.e21102 (2023). Harvey, J. J., Brant, S. R., Knutson, J. R. & Han, M. K. SNP analysis using CataCleave probes. J Clin Lab Anal 22 , 192-203, doi:10.1002/jcla.20240 (2008). Chen, Y., Strickland, M. R., Soranno, A. & Holtzman, D. M. Apolipoprotein E: Structural Insights and Links to Alzheimer Disease Pathogenesis. Neuron 109 , 205-221, doi:10.1016/j.neuron.2020.10.008 (2021). Nam, H. et al. PROMER technology: A new real-time PCR tool enabling multiplex detection of point mutation with high specificity and sensitivity. Biol Methods Protoc 9 , bpae041, doi:10.1093/biomethods/bpae041 (2024). Nam, Y. H. et al. A new quantitative real-time PCR method to measure human miRNAs using the PROMER technology. Biochem Biophys Res Commun 741 , 151069, doi:10.1016/j.bbrc.2024.151069 (2024). Zhong, L. et al. A rapid and cost-effective method for genotyping apolipoprotein E gene polymorphism. Mol Neurodegener 11 , 2, doi:10.1186/s13024-016-0069-4 (2016). Schaffer, S. et al. Variability in APOE genotype status in human-derived cell lines: a cause for concern in cell culture studies? Genes Nutr 9 , 364, doi:10.1007/s12263-013-0364-4 (2014). Tables Tables 1 to 6 are available in the Supplementary Files section Additional Declarations No competing interests reported. Supplementary Files Tables.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6415246","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":451116337,"identity":"3458654f-851e-4067-bb9b-b8d75b09f73b","order_by":0,"name":"Hyemin Kim","email":"","orcid":"","institution":"NuriBio Co., Ltd","correspondingAuthor":false,"prefix":"","firstName":"Hyemin","middleName":"","lastName":"Kim","suffix":""},{"id":451116343,"identity":"76cfc93c-a4fe-48f7-8201-122f9614764c","order_by":1,"name":"June Hahk Bae","email":"","orcid":"","institution":"Kangwon National 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00:53:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6415246/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6415246/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":81932867,"identity":"ff624421-a954-4ee9-9274-24b788a74b92","added_by":"auto","created_at":"2025-05-05 05:37:44","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":116042,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eApoE Gene Location and Its Alleles at Exon 4\u003c/strong\u003e\u003cbr\u003e\nThe ApoE gene is located on chromosome 19q13.2. Its alleles differ based on the amino acid positions at codons 112 and 158, resulting in six distinct genotypes.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6415246/v1/f0fbf0d4642827b9e252d09d.png"},{"id":81932871,"identity":"1d955ef3-b070-43e3-917b-070f830698b9","added_by":"auto","created_at":"2025-05-05 05:37:44","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":58304,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDesign of CataCleave Probes and Common Forward and Reverse Primers\u003c/strong\u003e\u003cbr\u003e\nFour different CataCleave probes were designed to specifically discriminate between the alleles at codons 112 and 158. Each probe was labeled with a distinct fluorescent reporter dye—FAM, HEX, Texas Red, or Cy5. A common forward and reverse primer pair was used to amplify the region encompassing these codons.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6415246/v1/91b17432897bac1b494e96ed.png"},{"id":81936124,"identity":"313d5f5b-851f-4722-8132-cdd1406a49ae","added_by":"auto","created_at":"2025-05-05 06:04:04","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":226128,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProof-of-Concept Genotyping Results Using Genomic DNA from Cell Lines\u003c/strong\u003e\u003cbr\u003e\nGenomic DNA from PC-3 (ε2/ε2), A549 (ε3/ε3), and U937 (ε4/ε4) cell lines, either individually or in combination, was analyzed by real-time quantitative PCR using the CataCleave probes along with the common forward and reverse primers.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6415246/v1/a21e1e55b72d2f3676837606.png"},{"id":81932870,"identity":"4e4561cc-cef7-4b26-baa1-ec8d4554b0e0","added_by":"auto","created_at":"2025-05-05 05:37:44","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":263406,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRepresentative Genotyping Results from Genomic DNA Isolated from Whole Blood and Buccal Swabs\u003c/strong\u003e\u003cbr\u003e\nGenomic DNA extracted from whole blood and buccal swabs of 50 healthy donors was genotyped using the CataCleave probes and common primer set. Four representative genotypes are shown as examples.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6415246/v1/54f6019a4f6b513e68baad6b.png"},{"id":99698566,"identity":"509ef8ea-abbe-47d2-bf04-ee446a6457dc","added_by":"auto","created_at":"2026-01-07 11:24:51","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1079061,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6415246/v1/3f7c3d8e-b90f-4230-9d5b-563f60157ec4.pdf"},{"id":81936123,"identity":"718b71db-990d-4d04-968f-a503503319c3","added_by":"auto","created_at":"2025-05-05 06:04:04","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":40504,"visible":true,"origin":"","legend":"","description":"","filename":"Tables.docx","url":"https://assets-eu.researchsquare.com/files/rs-6415246/v1/f715bdf257aa6c51538713d7.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"One pot PCR genotyping of ApoE for diagnosing Alzheimer’s disease","fulltext":[{"header":"Introduction","content":"\u003cp\u003eApolipoprotein E (apoE) genotyping is a critical tool for assessing genetic risk associated with Alzheimer\u0026rsquo;s disease (AD), particularly late-onset Alzheimer\u0026rsquo;s, the most common form of the condition \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. The \u003cem\u003eAPOE\u003c/em\u003e gene has three major alleles\u0026mdash;ε2, ε3, and ε4\u0026mdash;and each individual inherits one allele from each parent. These alleles influence the likelihood of developing AD. Notably, the ε4 allele is strongly associated with an increased risk and earlier onset of AD symptoms. Individuals carrying one copy (heterozygous) or two copies (homozygous) of ε4 face a significantly higher risk compared to those without the allele \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. In contrast, the ε2 allele may offer a protective effect, lowering the risk of disease development.\u003c/p\u003e \u003cp\u003eApoE genotyping is especially valuable for identifying individuals who may benefit from early monitoring or participation in clinical trials targeting high-risk populations \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. It also enhances our understanding of the biological mechanisms underlying AD, as apoE plays a role in amyloid-beta metabolism, lipid transport, and neuronal repair \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. While apoE genotyping is not a standalone diagnostic tool, it becomes particularly informative when combined with clinical evaluations, neuroimaging, and biomarker analyses. Together, these tools can improve risk assessment and support personalized prevention or intervention strategies \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eCurrent PCR-based methods for apoE genotyping typically require at least two reaction tubes\u0026mdash;and often more\u0026mdash;to distinguish among the six possible genotypes using combinations of allele-specific primers \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Consolidating the assay into a single-tube format would greatly reduce assay time, minimize manual labor, and improve throughput.\u003c/p\u003e \u003cp\u003eTo address this, we developed a one-pot PCR assay platform for apoE genotyping utilizing an RNA-DNA hybrid CataCleave probe. This probe is a chimeric DNA-RNA-DNA structure incorporating two internal fluorophores \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Upon hybridization to the target DNA, ribonuclease H (RNase H) cleaves the RNA portion of the probe, causing the resulting fragments to dissociate. This cleavage restores the donor fluorescence signal, allowing for precise and efficient detection of target sequences.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cem\u003eAssay Principle\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe APOE gene, located on chromosome 19q13.2, encodes a 299-amino acid apolipoprotein E protein, which exists in three major isoforms\u0026mdash;ApoE2, ApoE3, and ApoE4\u0026mdash;distinguished by two amino acid substitutions at positions 112 and 158 \u003csup\u003e8\u003c/sup\u003e. The codons TGC and CGC encode cysteine and arginine, respectively. Consequently, the isoforms differ by having either Cys or Arg at these positions (Fig. 1).\u003c/p\u003e\n\u003cp\u003eTo discriminate among the six possible apoE genotypes, we developed a panel of four CataCleave probes, each labeled with a unique 5\u0026prime; fluorescent reporter dye and a common 3\u0026prime; quencher. The probes were designated as follows: 112T (FAM), 112C (HEX), 158T (Texas Red), and 158C (Cy5). A single pair of common forward and reverse primers was designed to amplify the region encompassing these polymorphisms (Fig. 2). Each probe contains a single ribonucleotide (either rU or rC), positioned to hybridize with a complementary base (A or G) in the target DNA (Table 1). Upon perfect base pairing, the RNA-DNA hybrid is cleaved by RNase H2, resulting in probe fragmentation and fluorescence signal restoration. This enables real-time detection of allele-specific amplification.\u003c/p\u003e\n\u003cp\u003eTo validate the assay, genomic DNA was extracted from cell lines with known apoE genotypes. Homozygous genotypes\u0026mdash;\u0026epsilon;2/\u0026epsilon;2, \u0026epsilon;3/\u0026epsilon;3, and \u0026epsilon;4/\u0026epsilon;4\u0026mdash;were confirmed using PC3, A549, and U937 cells, respectively. Heterozygous genotypes were reconstructed by mixing DNA from two cell lines (Table 2, Fig. 3). All homozygous genotypes were successfully amplified with Ct values \u0026lt;25. Heterozygous genotypes were detected with Ct values \u0026lt;38. The assay produced distinct fluorescence signatures for each genotype. For example, \u0026epsilon;2/\u0026epsilon;2 generated FAM and Texas Red signals, while \u0026epsilon;2/\u0026epsilon;4 produced FAM, Texas Red, HEX, and Cy5 signals.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eLimit of Detection, Interference, and Specificity\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe analytical sensitivity of the assay was evaluated by performing eight replicate reactions per genotype using serial dilutions of genomic DNA. The limit of detection (LoD) ranged from 0.05 to 0.5 ng/\u0026mu;L (Table 3). To assess potential interference, representative serum components\u0026mdash;albumin, cholesterol, hemoglobin, and triglycerides\u0026mdash;were added to the reactions. None of these interfered with assay performance (Table 4). Assay specificity was evaluated by spiking the reaction mixtures with nucleic acids from 11 bacterial and viral species. No cross-reactivity or false-positive results were observed, confirming high specificity (Table 5). Reproducibility was assessed by performing the assay in duplicate for seven consecutive days and twice daily over a 20-day period. In all cases, the coefficient of variation (CV) was \u0026lt;5% (data not shown), demonstrating excellent precision and inter-assay consistency.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eClinical Sample Testing\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eFollowing analytical validation, the assay was applied to 50 clinical samples collected from healthy volunteers. Genomic DNA was isolated from both peripheral blood and buccal swabs. As shown in Fig. 4 and Table 6, genotype calls were fully concordant across both sample types, demonstrating the assay\u0026rsquo;s robustness and suitability for clinical applications. Four distinct genotypes were reliably identified in the test population.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn the present study, we developed a novel one-pot real-time quantitative PCR assay for apoE genotyping, utilizing our proprietary CataCleave probe technology. This platform, along with its enhanced version\u0026mdash;Promer technology\u0026mdash;enables sensitive and precise detection of single-point mutations, single nucleotide polymorphisms (SNPs), and microRNAs \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. The core principle of both technologies lies in the specific cleavage of RNA-DNA hybrids by RNase H2, which occurs only upon perfect sequence complementarity.\u003c/p\u003e \u003cp\u003eTo discriminate among the six possible apoE genotypes, we designed four distinct CataCleave probes\u0026mdash;each containing a strategically placed ribonucleotide\u0026mdash;and a shared pair of forward and reverse primers. Depending on the genotype of the sample, the probes can produce up to four distinct fluorescent signals. For example, the ε2/ε4 genotype yields four separate fluorescence channels, each corresponding to a specific allele-probe match.\u003c/p\u003e \u003cp\u003eSeveral commercial apoE genotyping kits are currently available; however, most employ two or more probe sets in separate reactions to independently assess the polymorphic positions at codons 112 and 158 of the \u003cem\u003eAPOE\u003c/em\u003e gene \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. While some newer products offer one-pot PCR solutions, the assay principles and supporting validation data have not been disclosed. In contrast, our study presents comprehensive details regarding the assay mechanism, limit of detection, specificity, lack of interference from common serum components, and assay precision. We believe this transparency will enable other laboratories to readily adopt our approach and perform accurate apoE genotyping in a single-tube format.\u003c/p\u003e \u003cp\u003eDespite the demonstrated reliability of our assay in clinical samples derived from both whole blood and buccal swabs, we did not observe any ε2/ε2 or ε4/ε4 homozygous genotypes in our sample set. This limitation likely reflects the relatively small sample size and the lower prevalence of these genotypes in the general population. Future studies involving larger and more diverse cohorts will be necessary to validate the assay's performance across all genotype categories and to better estimate the distribution of apoE alleles across different populations.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eReagents\u003c/h2\u003e \u003cp\u003eAll DNA and RNA oligonucleotides were synthesized by Integrated DNA Technologies (Coralville, IA, USA). RNase H2 was purchased from BioAssay Co. Ltd (Daejeon, South Korea) or produced in-house. Briefly, cDNA of RNase H2 (\u003cem\u003epyrococcus furiosus\u003c/em\u003e, GenBank: CP023154.1) was cloned into pET-28a plasmid and transformed into BL21(DE3)pLysS strain. Expression of the RNase H2 was induced with IPTG and purified using Ni-NTA affinity chromatography (ThermoFisher Scientific, Waltham, MA, USA) as previously reported \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCell culture\u003c/h3\u003e\n\u003cp\u003ePC-3, A549 and U937 human cell lines were purchased from Korea Cell Line Bank ( \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://cellbank.snu.ac.kr/main/\u003c/span\u003e\u003cspan address=\"http://cellbank.snu.ac.kr/main/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). They were cultured in DMEM culture media containing 10% FBS and 1% antibiotics at 37 and 5% CO\u003csub\u003e2\u003c/sub\u003e according to instruction provided by KCLB. They were sub-cultured at 80% confluency using trypsin/EDTA at 1:8 ratio.\u003c/p\u003e\n\u003ch3\u003eGenomic DNA isolation\u003c/h3\u003e\n\u003cp\u003eGenomic DNA was extracted from 2ⅹ10\u003csup\u003e6\u003c/sup\u003e cells, whole blood or buccal swabs by using the DNA extraction kit (Accuprep\u0026reg;, Bioneer, Korea) according to the manufacturer\u0026rsquo;s instructions. DNA was diluted with nuclease-free water to 10 ng/\u0026micro;L for apoE genotyping analysis.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eQuantitative real-time PCR\u003c/h2\u003e \u003cp\u003eApoE genotyping by Real Time PCR includes forward, reverse primer and four different kinds of CataCleave probes. Each PCR reaction mixture (15 \u0026micro;L) contained the following reagents: 1 \u0026times; genotyping master mix, 0.5 \u0026micro;M of forward primer and \u003cem\u003e112T, 112C, 158T, 158C\u003c/em\u003e probes, 0.5 \u0026micro;M of reverse primer, the indicated amounts of the genomic DNA. The PCR amplification protocol was as follows: Initial activation of nTaq DNA polymerase at 95\u0026deg;C for 5 min, followed by 40 cycles with denaturation at 95\u0026deg;C for 15 sec, and annealing/extension at 65\u0026deg;C for 1 min. The fluorescence signals were collected during the annealing/extension step. FAM, HEX, TexasRed and Cy5 signals were read by using the CFX96 system (Bio-Rad Laboratories, Hercules, CA, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eClinical samples\u003c/h2\u003e \u003cp\u003eWhole blood and buccal swabs were collected from 50 healthy volunteers. Informed consent was obtained by Shihwa Medical Center, and the study was approved by the Institutional Review Board (Shihwa 2022\u0026thinsp;\u0026minus;\u0026thinsp;0124). All methods were performed in accordance with the relevant guidelines and regulations. After collecting blood sample, it was stored for up to 1 hour at room temperature (15\u0026thinsp;~\u0026thinsp;30℃) or up to 3 days at 2\u0026thinsp;~\u0026thinsp;10℃. For long-term storage, it was stored for up to one month if stored at -70℃ or below. For buccal swabs, it was stored in a transport medium for nucleic acid preservation for up to one month at 2\u0026thinsp;~\u0026thinsp;8 ℃.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eCoefficient of variance (CV) were calculated using Excel software.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eKim H, Yang M, Yang Y, Cha W, Kwak T, Jeon S, Bae S, Son S, Lee E, and Yang H performed the experiments and collected the data. Bae JH and Nam YH designed the experiment and supervised the study. Shin JS analyzed the data and wrote the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors declare no potential conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.\u003cbr\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eRaulin, A. C.\u003cem\u003e et al.\u003c/em\u003e ApoE in Alzheimer\u0026apos;s disease: pathophysiology and therapeutic strategies. \u003cem\u003eMol Neurodegener\u003c/em\u003e \u003cstrong\u003e17\u003c/strong\u003e, 72, doi:10.1186/s13024-022-00574-4 (2022).\u003c/li\u003e\n\u003cli\u003eLeoni, V. The effect of apolipoprotein E (ApoE) genotype on biomarkers of amyloidogenesis, tau pathology and neurodegeneration in Alzheimer\u0026apos;s disease. \u003cem\u003eClin Chem Lab Med\u003c/em\u003e \u003cstrong\u003e49\u003c/strong\u003e, 375-383, doi:10.1515/CCLM.2011.088 (2011).\u003c/li\u003e\n\u003cli\u003eKim, J., Basak, J. M. \u0026amp; Holtzman, D. M. The role of apolipoprotein E in Alzheimer\u0026apos;s disease. \u003cem\u003eNeuron\u003c/em\u003e \u003cstrong\u003e63\u003c/strong\u003e, 287-303, doi:10.1016/j.neuron.2009.06.026 (2009).\u003c/li\u003e\n\u003cli\u003eHuang, Y. \u0026amp; Mahley, R. W. Apolipoprotein E: structure and function in lipid metabolism, neurobiology, and Alzheimer\u0026apos;s diseases. \u003cem\u003eNeurobiol Dis\u003c/em\u003e \u003cstrong\u003e72 Pt A\u003c/strong\u003e, 3-12, doi:10.1016/j.nbd.2014.08.025 (2014).\u003c/li\u003e\n\u003cli\u003eLumsden, A. L., Mulugeta, A., Zhou, A. \u0026amp; Hypponen, E. Apolipoprotein E (APOE) genotype-associated disease risks: a phenome-wide, registry-based, case-control study utilising the UK Biobank. \u003cem\u003eEBioMedicine\u003c/em\u003e \u003cstrong\u003e59\u003c/strong\u003e, 102954, doi:10.1016/j.ebiom.2020.102954 (2020).\u003c/li\u003e\n\u003cli\u003eNajd-Hassan-Bonab, L., Hedayati, M., Shahzadeh Fazeli, S. A. \u0026amp; Daneshpour, M. S. An optimized method for PCR-based genotyping to detect human APOE polymorphisms. \u003cem\u003eHeliyon\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, e21102, doi:10.1016/j.heliyon.2023.e21102 (2023).\u003c/li\u003e\n\u003cli\u003eHarvey, J. J., Brant, S. R., Knutson, J. R. \u0026amp; Han, M. K. SNP analysis using CataCleave probes. \u003cem\u003eJ Clin Lab Anal\u003c/em\u003e \u003cstrong\u003e22\u003c/strong\u003e, 192-203, doi:10.1002/jcla.20240 (2008).\u003c/li\u003e\n\u003cli\u003eChen, Y., Strickland, M. R., Soranno, A. \u0026amp; Holtzman, D. M. Apolipoprotein E: Structural Insights and Links to Alzheimer Disease Pathogenesis. \u003cem\u003eNeuron\u003c/em\u003e \u003cstrong\u003e109\u003c/strong\u003e, 205-221, doi:10.1016/j.neuron.2020.10.008 (2021).\u003c/li\u003e\n\u003cli\u003eNam, H.\u003cem\u003e et al.\u003c/em\u003e PROMER technology: A new real-time PCR tool enabling multiplex detection of point mutation with high specificity and sensitivity. \u003cem\u003eBiol Methods Protoc\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, bpae041, doi:10.1093/biomethods/bpae041 (2024).\u003c/li\u003e\n\u003cli\u003eNam, Y. H.\u003cem\u003e et al.\u003c/em\u003e A new quantitative real-time PCR method to measure human miRNAs using the PROMER technology. \u003cem\u003eBiochem Biophys Res Commun\u003c/em\u003e \u003cstrong\u003e741\u003c/strong\u003e, 151069, doi:10.1016/j.bbrc.2024.151069 (2024).\u003c/li\u003e\n\u003cli\u003eZhong, L.\u003cem\u003e et al.\u003c/em\u003e A rapid and cost-effective method for genotyping apolipoprotein E gene polymorphism. \u003cem\u003eMol Neurodegener\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 2, doi:10.1186/s13024-016-0069-4 (2016).\u003c/li\u003e\n\u003cli\u003eSchaffer, S.\u003cem\u003e et al.\u003c/em\u003e Variability in APOE genotype status in human-derived cell lines: a cause for concern in cell culture studies? \u003cem\u003eGenes Nutr\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 364, doi:10.1007/s12263-013-0364-4 (2014).\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 to 6 are available in the Supplementary Files section\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"ApoE genotype, Real-time PCR, Alzheimer’s disease, CataCleave probe","lastPublishedDoi":"10.21203/rs.3.rs-6415246/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6415246/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eApolipoprotein E (apoE) genotyping is a valuable tool for assessing genetic risk associated with Alzheimer\u0026rsquo;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.\u003c/p\u003e \u003cp\u003eMany studies have reported the use of PCR-based methods for apoE genotyping. However, current approaches typically require at least two tubes\u0026mdash;and often more\u0026mdash;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.\u003c/p\u003e \u003cp\u003eTo 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\u0026rsquo;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.\u003c/p\u003e","manuscriptTitle":"One pot PCR genotyping of ApoE for diagnosing Alzheimer’s disease","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-05 05:37:39","doi":"10.21203/rs.3.rs-6415246/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"517954b4-70ec-467c-8179-964a972852c1","owner":[],"postedDate":"May 5th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":47996272,"name":"Biological sciences/Biological techniques/Genetic techniques/Pcr based techniques"},{"id":47996273,"name":"Health sciences/Diseases/Neurological disorders/Dementia/Alzheimers disease"}],"tags":[],"updatedAt":"2026-01-07T11:23:25+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-05 05:37:39","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6415246","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6415246","identity":"rs-6415246","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}