Whole-genome amplified DNA from stored dried blood spots is reliable in high resolution melting curve and sequencing analysis.
Whole-genome amplified DNA from stored dried blood spots is reliable in high resolution melting curve and sequencing analysis.
复制标题
DOI：
10.1186/1471-2350-12-22
复制
发表时间：
2011-02-09
影响因子：
--
通讯作者：
Tfelt-Hansen J
中科院分区：
文献类型：
--
作者：
Winkel BG;Hollegaard MV;Olesen MS;Svendsen JH;Haunsø S;Hougaard DM;Tfelt-Hansen J
干血斑（DBS）样本在基因组检查中的使用受到其所含基因组DNA（gDNA）相对较低的限制。来自储存的DBS样品的全基因组扩增DNA（wgaDNA）构成gDNA的可靠替代物，这仍有待证明。我们想要比较来自DBS样品的wgaDNA与来自全血的gDNA的解链曲线和测序结果。从10名孤立性房颤患者（平均年龄22.3岁）的全血中提取gDNA。从储存在-24 ° C的新生DBS样品中提取基因组DNA，并一式三份扩增全基因组。在wgaDNA和gDNA样品中使用高分辨率解链曲线分析和直接测序，研究了基因SCN 5A和KCNA 5的所有编码区和相邻内含子区。在85个wgaDNA样品和81个gDNA样品中存在改变的解链曲线。序列分析在10个wgaDNA样品中鉴定了总共31个变体。在gDNA组中的完全相同的样本模式中发现了相同的31个变体。在wgaDNA组中不存在假阳性或假阴性序列变异。从DBS样品中一式三份扩增的DNA的使用是可靠的，并且可用于高分辨率曲线解链分析以及直接序列分析。因此，DBS样本可以作为全血序列分析的替代品。
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.
登录
查看更多内容
影响因子：
3.9
作者：
Dobrowolski, SE;McKinney, JT;Longo, N
通讯作者：
Longo, N
影响因子：
6.7
作者：
Simard, C;Drolet, B;Roden, DM
通讯作者：
Roden, DM
影响因子：
5.2
作者：
Gouas, L;Nicaud, V;Guicheney, P
通讯作者：
Guicheney, P
DOI：
10.1006/bmme.1996.0017
发表时间：
1996-04-01
期刊：
BIOCHEMICAL AND MOLECULAR MEDICINE
影响因子：
--
作者：
Therrell, BL;Hannon, WH;Shapira, E
通讯作者：
Shapira, E
影响因子：
4.5
作者：
Wilcken, Bridget;Wiley, Veronica
通讯作者：
Wiley, Veronica