Large-scale Extraction of DNA by Using Salting-out Principle for Dried Blood Spots to Screen Multiple Mutations in GCDH Gene

Dried blood spots (DBS) are the best sampling for large and population-based genetic studies. The study aimed to develop a DNA extraction protocol for DBS samples in a 96-well microtiter plate format using DNA salting-out principle. The developed protocol was named High-Throughput Salting-Out (HTSO). The DBS samples were collected from 20 suspected Glutaric Acidemia Type I (GA-I) patients from unrelated families and 50 controls without any metabolic disorders. The DNA was isolated from DBS samples using HTSO protocol in a 96-well microtiter plate. The assessment of yield and quality of DNA after isolation by HTSO method was done using NanoDrop-8000. The DNA isolated by the HTSO was used in the genotyping of multiple mutations, R402W and W225X in the Glutaryl-CoA Dehydrogenase gene in GA-I patients by multiplex PCR and RFLP. The HTSO showed at least a 2- to 34-fold increased DNA yield against manual in-house, commercial, and automation-based protocols. The per sample DNA extraction cost of the HTSO method was four times lesser than that of the commercial kits. Without sophisticated instruments or automation, HTSO method considerably reduced the turnaround time of DNA extraction compared to manual in-house and commercial kit protocols. The HTSO method is efficient, inexpensive, and time-saving for large-scale and potential high-throughput DNA extraction for DBS samples. This method is adequate for high-end diagnostic and institution facilities, in addition to institutions having financial constraints or lacks sophisticated instruments.