Gerber Livia, Whiteley Sarah L, Hahn Erin E, Holleley Clare E
Australian National Wildlife Collection CSIRO, National Research Collections Australia, Black Mountain, ACT, Australia.
Institute for Applied Ecology, University of Canberra, Bruce, ACT, Australia.
PLoS One. 2025 Jul 31;20(7):e0329019. doi: 10.1371/journal.pone.0329019. eCollection 2025.
Specialized chemically-coated paper cards, such as Flinders Technology Associates (FTA) cards, provide simple and reliable storage of nucleic acids by protecting DNA from degradation. Owed to their simplicity, FTA cards are widely used in clinical testing, forensic science and specimen archives. Originally developed for PCR-based applications that only require short DNA fragments, FTA cards are now being explored as an avenue for whole-genome and epigenetic sequencing applications. FTA cards and their corresponding DNA extraction protocols have not kept pace with advances in sequencing technologies. Because the initial protocols developed for FTA cards were geared towards applications using PCR amplification of short fragments, they typically yield low molecular weight DNA. This issue is particularly pronounced for FTA elute cards where heat-based elution at 95°C leads to DNA denaturation and fragmentation. Isolation of DNA from nucleated blood deposited onto FTA elute cards poses an additional challenge when compared to FTA classic cards, because hemoglobin is irreversibly bound to the card matrix, making the majority of DNA present in nucleated blood inaccessible. Here, we describe an easy, fast, and inexpensive protocol to extract high molecular weight DNA (>10 kb) of nucleated blood stored on FTA elute cards suitable for most genomic library preparations including those that interrogate DNA methylation. Our protocol yields a 14-fold increase in yield compared to numerous alternatives. Using our protocol, we demonstrate that high molecular weight DNA can still be extracted even after storage at ambient temperature for over a decade. Moreover, we show that DNA methylation marks are preserved on FTA elute cards, broadening the utility of FTA elute cards. This opens possibilities for (epi-)genomic studies using historical samples and enabling specimen collection where access to chemicals or cryogenic storage is limited - reducing project costs and extending collection opportunities into remote areas.
特殊的化学涂层纸卡，如弗林德斯技术协会（FTA）卡，通过保护DNA不被降解，提供了简单可靠的核酸储存方式。由于其操作简便，FTA卡广泛应用于临床检测、法医学和标本存档。FTA卡最初是为仅需要短DNA片段的基于PCR的应用而开发的，现在正被探索作为全基因组和表观遗传测序应用的一种途径。FTA卡及其相应的DNA提取方案并未跟上测序技术的发展。因为最初为FTA卡开发的方案是针对使用短片段PCR扩增的应用，它们通常产生低分子量的DNA。对于FTA洗脱卡来说，这个问题尤为突出，因为在95°C下基于加热的洗脱会导致DNA变性和片段化。与FTA经典卡相比，从沉积在FTA洗脱卡上的有核血中分离DNA带来了额外的挑战，因为血红蛋白与卡基质不可逆地结合，使得有核血中存在的大部分DNA无法获取。在这里，我们描述了一种简单、快速且廉价的方案，用于从存储在FTA洗脱卡上的有核血中提取高分子量DNA（>10 kb），适用于大多数基因组文库制备，包括那些检测DNA甲基化的制备。与众多其他方案相比，我们的方案产量提高了14倍。使用我们的方案，我们证明即使在室温下储存十多年后，仍可提取高分子量DNA。此外，我们表明FTA洗脱卡上的DNA甲基化标记得以保留，拓宽了FTA洗脱卡的用途。这为使用历史样本进行（表观）基因组研究开辟了可能性，并使得在获取化学品或低温储存受限的地方能够进行样本采集——降低项目成本并将采集机会扩展到偏远地区。
