Abstracts

Rationale: Genetic testing and research have increased demand for high quality DNA for downstream applications. However, infants, control population, and SUDEP cases present unique challenges in the traditional invasive collection of the biological specimen by venipuncture. Guthrie/FTA cards based blood spots, buccal scrapes and finger nail clippings are attractive, alternative DNA sources. The literature details a variety of protocols for extraction of nucleic acids from these tissues. The universally reported endpoint of a successful procedure is the overall yield but not the performance of the DNA in methods commonly used in mutation detection. Here we performed a direct, systematic comparison of several biological specimens and extraction protocols, evaluating not only the final DNA quantity and quality, but also its utility in downstream genetic applications.Methods: Blood spots, buccal scrapes and fingernail clippings were collected and processed within one week (buccal scrapes and fingernails) or at various time points (blood spots) to mimic prospective procurement vs. a retrospective pull of an infantile screening card for SUDEP cases. Detailed literature search identified the most prevalent commercial and homemade extraction protocols that were systematically applied to process the collected specimens. The endpoints of DNA quality, quantity, and utility were compared across sample types and protocols.Results: We identified three distinct stages of extraction protocols with a direct impact on the resulting DNA yield, quality, and performance; 1) washing 2) extraction, and 3) purification. This information led to the generation of an optimized universal protocol for reliable extraction of DNA with an optimal, source dependent yield and quality from all sample types evaluated in our study. Fingernail-derived samples yielded high DNA quantity 61+/-21ng/ul while the best quality DNA was obtained from dried blood spots where the sample age inversely affected the yield (14+/-1.2ng/ul in samples processed after three weeks vs. 7+/-0.9ng/ul in specimens extracted on the day of collection). The DNA performance was evaluated by PCR which generated exome specific amplicons up to 1 kb in length. They performed optimally in the subsequent SNP detection by Sanger sequencing regardless of the original specimen thus demonstrating the utility of these alternative DNA tissue sources in epilepsy genetic research and testing.Conclusions: Fingernail trimmings, Guthrie/FTA card stabilized blood spots, and buccal scrapes are valuable alternative DNA sources. Their collection, storage, and distribution are economical, amenable to large scale efforts, and require minimal or no expertise. Their particular utility is in pediatric, retrospective, and post-mortem genetic analyses. Our investigation outlines the biological specimen handling and universal extraction protocol that ensures DNA of sufficient quality and quantity for downstream genetic applications. Grants: NS067013-01A1,CURE(AMG), NS-049130,Blue Bird Circle Foundation(JLN), Deutsche Forschungsgemeinschaft GSN 82/1(ELR)