Dept. of Cardiology, Rigshospitalet and Danish National Research Foundation Centre for Cardiac Arrhythmia (DARC), Copenhagen, Denmark.
BMC Med Genet. 2011 Feb 9;12:22. doi: 10.1186/1471-2350-12-22.
The use of dried blood spots (DBS) samples in genomic workup has been limited by the relative low amounts of genomic DNA (gDNA) they contain. It remains to be proven that whole genome amplified DNA (wgaDNA) from stored DBS samples, constitutes a reliable alternative to gDNA.We wanted to compare melting curves and sequencing results from wgaDNA derived from DBS samples with gDNA derived from whole blood.
gDNA was extracted from whole blood obtained from 10 patients with lone atrial fibrillation (mean age 22.3 years). From their newborn DBS samples, stored at -24°C, genomic DNA was extracted and whole-genome amplified in triplicates. Using high resolution melting curve analysis and direct sequencing in both wgaDNA and gDNA samples, all coding regions and adjacent intron regions of the genes SCN5A and KCNA5 were investigated.
Altered melting curves was present in 85 of wgaDNA samples and 81 of gDNA samples. Sequence analysis identified a total of 31 variants in the 10 wgaDNA samples. The same 31 variants were found in the exact same pattern of samples in the gDNA group. There was no false positive or negative sequence variation in the wgaDNA group.
The use of DNA amplified in triplicates from DBS samples is reliable and can be used both for high resolution curve melting analysis as well as direct sequence analysis. DBS samples therefore can serve as an alternative to whole blood in sequence analysis.
由于干血斑（DBS）样本中所含的基因组 DNA（gDNA）相对较少，因此其在基因组研究中的应用受到限制。需要证明从储存的 DBS 样本中获得的全基因组扩增 DNA（wgaDNA）可以替代 gDNA。我们希望比较从 DBS 样本中获得的 wgaDNA 与从全血中获得的 gDNA 的熔解曲线和测序结果。
从 10 名孤立性心房颤动患者（平均年龄 22.3 岁）的全血中提取 gDNA。从他们储存于-24°C 的新生儿 DBS 样本中提取基因组 DNA，并进行三次全基因组扩增。使用高分辨率熔解曲线分析和直接测序，对 SCN5A 和 KCNA5 基因的所有编码区和相邻内含子区进行研究。
在 85 个 wgaDNA 样本和 81 个 gDNA 样本中存在异常熔解曲线。序列分析在 10 个 wgaDNA 样本中总共鉴定出 31 种变体。在 gDNA 组中，相同的 31 种变体以完全相同的模式出现在相同的样本中。在 wgaDNA 组中没有假阳性或假阴性的序列变异。
使用 DBS 样本中经三次扩增的 DNA 进行可靠的研究，既可以用于高分辨率曲线熔解分析，也可以用于直接序列分析。因此，DBS 样本可以替代全血进行序列分析。
