Vásquez Bonilla Maria Mercedes, Guerrero-Freire Mónica Salome, Ledesma Yanua, Laglaguano Juan Carlos, de Waard Jacobus H
One Health Research Group, Facultad de Ciencias de la Salud, Universidad de Las Américas, Quito, 170124, Ecuador.
Programa de Doctorado, Facultad de Ciencias Veterinarias, Universidad de Buenos Aires, Buenos Aires, C1063ACV, Argentina.
Biol Methods Protoc. 2024 Oct 26;9(1):bpae079. doi: 10.1093/biomethods/bpae079. eCollection 2024.
We present a rapid high-throughput DNA extraction method for use with EDTA-anticoagulated blood using silicon dioxide (SiO) powder in a guanidine-HCl solution, hereinafter referred to as "Glassmilk." The method utilizes a 96-well deep-well plate, enabling DNA extraction from 96 samples in under 3 h. The method integrates cell lysis, washing, elution, and DNA storage within the same well, eliminating the need for DNA transfer. The Glassmilk extraction method is cost-effective and fast, and it avoids expensive or toxic reagents by using only basic lab equipment. The method yielded approximately 40 μg of high-quality DNA from 200 μl of blood. The DNA yield of the Glassmilk method was about 50% higher, and the purity of the DNA was comparable to those obtained using two commercial column-based extraction kits that were used for comparison. The cost per sample was around $1, with the most expensive item being the filter pipette tips, which account for about $0.80 per sample. As we show, the extracted DNA is suitable for downstream applications such as polymerase chain reaction (PCR), PCR-restriction fragment length polymorphism analysis, and qPCR. The method can be adapted for various sample types, including biopsies, fecal samples, cultured cells, and bacteria (see "subprotocols" section), and can also be applied in individual Eppendorf tubes. Our protocol may be useful for basic molecular research in laboratories having limited funds.
我们展示了一种快速高通量的DNA提取方法，该方法使用盐酸胍溶液中的二氧化硅（SiO）粉末（以下简称“玻璃奶”）从乙二胺四乙酸（EDTA）抗凝血液中提取DNA。该方法采用96孔深孔板，能够在3小时内从96个样本中提取DNA。该方法在同一孔内整合了细胞裂解、洗涤、洗脱和DNA储存步骤，无需转移DNA。玻璃奶提取方法经济高效且快速，仅使用基本的实验室设备，避免了使用昂贵或有毒的试剂。该方法从200μl血液中可获得约40μg高质量的DNA。玻璃奶方法的DNA产量高出约50%，DNA纯度与用于比较的两种基于柱的商业提取试剂盒相当。每个样本的成本约为1美元，其中最贵的物品是过滤吸头，每个样本约占0.80美元。如我们所示，提取的DNA适用于下游应用，如聚合酶链反应（PCR）、PCR限制性片段长度多态性分析和定量PCR。该方法可适用于各种样本类型，包括活检组织、粪便样本、培养细胞和细菌（见“子方案”部分），也可应用于单个Eppendorf管中。我们的方案可能对资金有限的实验室进行基础分子研究有用。
