Van Biesen Natalja, Cools Piet, Meyers Eline
Department of Diagnostic Sciences, Faculty of Medicine and Health Sciences, Ghent University, 9000 Ghent, Belgium.
Pediatr Rep. 2025 Mar 4;17(2):30. doi: 10.3390/pediatric17020030.
BACKGROUND/OBJECTIVES: DNA extraction from dried blood spot (DBS) samples is often applied in neonatal screening programs. Although various methods to extract DNA from DBSs have been described, the optimal approach remains unclear. Therefore, this study aimed to compare and optimize extraction methods to establish a reliable and efficient protocol for human DNA extraction from DBSs.
We conducted a back-to-back comparison of five different DNA extraction methods on 20 DBS samples: three column-based kits (QIAamp DNA mini kit, High Pure PCR Template Preparation kit, DNeasy Blood & Tissue kit) and two in-house boiling methods (one using TE buffer, one using Chelex-100 resin). DNA recovery was measured with DeNovix DS-11 and qPCR. Further optimization of elution volumes and starting material was performed on the best-performing methods (sample size = 5). Additionally, T-cell receptor excision circle (TREC) DNA was assessed by qPCR as an application.
The Chelex boiling method yielded significantly ( < 0.0001) higher DNA concentrations compared to the other methods. Column-based methods showed low DNA recovery, except for Roche, which showed significantly ( < 0.0001) higher DNA concentrations than the other column-based methods, as measured by DeNovix DS-11. Decreasing elution volumes (150 vs. 100 vs. 50 µL) increased DNA concentrations significantly, while increasing starting material (two vs. one 6 mm spot) did not.
We identified an easy and cost-effective optimized DNA extraction method using Chelex from DBSs, with an elution volume of 50 µL and 1 × 6 mm DBS punch, which is particularly advantageous for research in low-resource settings and large populations, such as neonatal screening programs.
背景/目的：从干血斑（DBS）样本中提取DNA常用于新生儿筛查项目。尽管已有多种从DBS中提取DNA的方法，但最佳方法仍不明确。因此，本研究旨在比较和优化提取方法，以建立一种可靠且高效的从DBS中提取人类DNA的方案。
我们对20个DBS样本的五种不同DNA提取方法进行了背对背比较：三种基于柱式的试剂盒（QIAamp DNA微量试剂盒、高纯PCR模板制备试剂盒、DNeasy血液与组织试剂盒）和两种自制煮沸法（一种使用TE缓冲液，一种使用Chelex-100树脂）。使用DeNovix DS-11和qPCR测量DNA回收率。对性能最佳的方法（样本量 = 5）进一步优化洗脱体积和起始材料。此外，通过qPCR评估T细胞受体切除环（TREC）DNA作为一种应用。
与其他方法相比，Chelex煮沸法产生的DNA浓度显著更高（<0.0001）。基于柱式的方法显示DNA回收率较低，除了罗氏试剂盒，通过DeNovix DS-11测量，其显示的DNA浓度比其他基于柱式的方法显著更高（<0.0001）。降低洗脱体积（150 μL对100 μL对50 μL）显著提高了DNA浓度，而增加起始材料（两个对一个6 mm血斑）则没有。
我们确定了一种使用Chelex从DBS中提取DNA的简便且经济高效的优化方法，洗脱体积为50 μL，使用1个6 mm的DBS冲孔，这对于资源匮乏地区和大量人群的研究（如新生儿筛查项目）特别有利。
