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Direct single-nucleotide polymorphism genotyping from whole blood without DNA extraction.
Abstract: A subset of single-nucleotide polymorphisms (SNPs) are associated with various phenotypes, including diseases, coat colour, and athletic performance, and are widely used in medical and veterinary fields. In this study, we developed a novel method for direct SNP genotyping from whole blood without DNA extraction. Thoroughbred blood samples were diluted 100-fold with Milli-Q water and analysed using real-time polymerase chain reaction (PCR) with hydrolysis probes. Specificity and sensitivity were improved by increasing the annealing temperature and number of PCR cycles. Genotyping results for SNPs in and showed complete concordance with conventional real-time PCR using the extracted DNA. This method is simple, low-cost, highly versatile, and applicable to other genetic targets, such as and .
©2026 The Japanese Society of Equine Science.
Publication Date: 2026-03-14 PubMed ID: 41859758PubMed Central: PMC12995552DOI: 10.1294/jes.37.35Google Scholar: Lookup The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
- Journal Article
Summary
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Overview
- This research presents a new technique for identifying single-nucleotide polymorphisms (SNPs) directly from whole blood samples without the need for DNA extraction.
- The method simplifies the genotyping process, making it cost-effective and broadly applicable, particularly in medical and veterinary genetics.
Background
- SNPs are variations at a single nucleotide position in the genome and are linked to various phenotypes such as diseases, coat colors in animals, and athletic performance.
- Genotyping SNPs is essential for medical diagnostics, breeding programs, and research into genetic traits.
- Traditional SNP genotyping requires DNA extraction from blood samples, which can be time-consuming and costly.
Research Objective
- To develop a method that allows SNP genotyping directly from whole blood, bypassing the conventional DNA extraction step.
- The goal was to maintain or improve the specificity and sensitivity of SNP detection while simplifying and reducing the overall costs and time involved.
Methodology
- Whole blood samples from Thoroughbred horses were diluted 100-fold using Milli-Q water.
- These diluted samples were subjected to real-time polymerase chain reaction (PCR) using hydrolysis probes specific for targeted SNPs.
- The research team optimized the PCR conditions by raising the annealing temperature and increasing the number of PCR cycles to enhance test specificity and sensitivity.
- Several SNP targets were selected for genotyping to test the method’s accuracy and reliability.
Results
- The direct genotyping from diluted whole blood showed complete concordance with the results obtained from conventional real-time PCR performed on extracted DNA samples.
- This demonstrated that skipping the DNA extraction did not compromise the accuracy of SNP genotyping.
- The enhanced PCR conditions facilitated reliable detection despite the presence of potential inhibitors in whole blood.
Advantages and Implications
- The method simplifies the genotyping workflow by eliminating DNA extraction, reducing labor and reagent costs.
- It is versatile, applicable not only to SNPs tested here but potentially to other genetic targets.
- Its simplicity and cost-effectiveness make it attractive for large-scale clinical and veterinary applications where rapid and reliable genetic screening is required.
Potential Applications
- Medical diagnostics for disease-related SNP markers.
- Genetic screening in veterinary medicine, including coat color determination and athletic trait analysis in horses and other animals.
- Population genetics and breeding programs where rapid genotyping is beneficial.
Summary
- This study successfully demonstrated a streamlined, direct SNP genotyping method from whole blood without DNA extraction using real-time PCR with hydrolysis probes.
- The approach maintained high accuracy, sensitivity, and specificity by optimizing PCR conditions.
- This innovation promises to make SNP genotyping more accessible and cost-effective across diverse research and clinical settings.
Cite This Article
APA
Furukawa R, Tozaki T, Kawate K, Kikuchi M, Ishige T, Fukui E, Kakoi H.
(2026).
Direct single-nucleotide polymorphism genotyping from whole blood without DNA extraction.
J Equine Sci, 37(1), 35-40.
https://doi.org/10.1294/jes.37.35 Publication
Researcher Affiliations
- Genetic Analysis Department, Laboratory of Racing Chemistry, Utsunomiya, Tochigi 320-0851, Japan.
- Genetic Analysis Department, Laboratory of Racing Chemistry, Utsunomiya, Tochigi 320-0851, Japan.
- Genetic Analysis Department, Laboratory of Racing Chemistry, Utsunomiya, Tochigi 320-0851, Japan.
- Genetic Analysis Department, Laboratory of Racing Chemistry, Utsunomiya, Tochigi 320-0851, Japan.
- Genetic Analysis Department, Laboratory of Racing Chemistry, Utsunomiya, Tochigi 320-0851, Japan.
- School of Agriculture, Utsunomiya University, Utsunomiya, Tochigi 321-8505, Japan.
- Genetic Analysis Department, Laboratory of Racing Chemistry, Utsunomiya, Tochigi 320-0851, Japan.
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