Comparison and Optimization of DNA Extraction Methods for Human DNA from Dried Blood Spot Samples.

Abstract

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.

Methods: 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 ACTB 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.

Results: The Chelex boiling method yielded significantly (p < 0.0001) higher ACTB DNA concentrations compared to the other methods. Column-based methods showed low DNA recovery, except for Roche, which showed significantly (p < 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 ACTB DNA concentrations significantly, while increasing starting material (two vs. one 6 mm spot) did not.

Conclusions: 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.