Newborn Screening Ontario, Ottawa, Ontario K1H 8L1, Canada.
Department of Pediatrics, University of Ottawa , Ottawa, Ontario K1H8M5, Canada.
Anal Chem. 2018 Jan 2;90(1):801-806. doi: 10.1021/acs.analchem.7b03265. Epub 2017 Dec 15.
Newborn screening programs have expanded to include molecular-based assays as first-tier tests and the success of these assays depends on the quality and yield of DNA extracted from neonatal dried blood spots (DBS). To meet high throughput and rapid turnaround time requirements, newborn screening laboratories adopted rapid DNA extraction methods that produce crude extracts. Quantification of DNA in neonatal DBS is not routinely performed due to technical challenges; however, this may enhance the performance of assays that are sensitive to amounts of input DNA. In this study, we developed a novel high throughput method to quantify total DNA in DBS. It is based on specific acid-catalyzed depurination of DNA followed by mass spectrometric quantification of adenine. The amount of adenine was used to calculate DNA quantity per 3.2 mm DBS. Reference intervals were established using archived, neonatal DBS (n = 501) and a median of 130.6 ng of DNA per DBS was obtained, which is in agreement with literature values. The intra- and interday variations were <15%. The limits of detection and quantification were 12.5 and 37.8 nmol/L adenine, respectively. We demonstrated that DNA from neonatal DBS can be successfully quantified in high throughput settings using instruments currently deployed in NBS laboratories.
新生儿筛查项目已经扩展到包括基于分子的检测作为第一线检测，这些检测的成功取决于从新生儿干血斑（DBS）中提取的 DNA 的质量和产量。为了满足高通量和快速周转时间的要求，新生儿筛查实验室采用了产生粗提取物的快速 DNA 提取方法。由于技术挑战，新生儿 DBS 中的 DNA 定量通常不进行;然而，这可能会增强对输入 DNA 量敏感的检测的性能。在这项研究中，我们开发了一种新的高通量方法来定量 DBS 中的总 DNA。它基于特定的酸催化的 DNA 脱嘌呤作用，随后进行腺嘌呤的质谱定量。腺嘌呤的量用于计算每个 3.2 毫米 DBS 的 DNA 量。使用存档的新生儿 DBS（n = 501）建立了参考区间，每个 DBS 获得的 DNA 中位数为 130.6 纳克，与文献值一致。日内和日间变化<15%。检测限和定量限分别为 12.5 和 37.8 nmol/L 腺嘌呤。我们证明，使用目前在 NBS 实验室中部署的仪器，可以在高通量环境中成功定量新生儿 DBS 中的 DNA。
