Optimization of Chelex 100 resin-based extraction of genomic DNA from dried blood spots

摘要

Dried blood spots (DBS) are widely utilized as part of universal newborn screening and as a means of transporting samples from field sites. We use DBS from African field sites to assess for rare maternal-fetal cell exchange during pregnancy known as microchimerism. We aimed to develop a protocol to maximize the quantity of high-quality genomic DNA (gDNA) extracted from DBS. The total gDNA yield obtained from control DBS utilizing a Qiagen-based protocol and a Chelex 100 resin-based protocol was first compared. Variations of the Chelex protocol were subsequently tested to develop an optimized protocol. The gDNA was quantified by qPCR targeting the human beta-globin gene. DNA yield for a given experimental condition was normalized to a Chelex control performed on the same day, and the total yields were compared using a Student's t-test. The control Chelex protocol yielded 590% more DNA than the QIAamp DNA Blood Mini Kit. The absolute efficiency of the control Chelex protocol was 54%, compared to an absolute efficiency of 9% for the QIAamp DNA Blood Mini Kit. Modification of the Chelex protocol to include a second heat precipitation from the same DBS increased the gDNA yield by 29% (P0.001). Our optimized protocol including this modification increased the absolute efficiency of extraction to 68%. The gDNA extracted using the Chelex protocol was stable through repeated freeze-thaw cycles. In a mock microchimerism experiment, rare donor alleles at a frequency of 10 in 100 000 could be identified in gDNA from DBS extracted using the optimized Chelex protocol. Our findings may be of significance for a diverse range of applications that utilize DBS and require high-quality DNA, including newborn screening programs, pathogen and drug resistance screening from remote field sites, forensics, and rare allele detection.