Toward a fluorescent single-strand conformation polymorphism technique that detects all mutations: F-DOVAM-S.
Toward a fluorescent single-strand conformation polymorphism technique that detects all mutations: F-DOVAM-S.
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开发可检测所有突变的荧光单链构象多态性技术：F-DOVAM-S。
DOI：
10.1016/j.ab.2007.05.024
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发表时间：
2007
影响因子：
2.9
通讯作者：
Sommer,SteveS
中科院分区：
文献类型：
--
作者：
Mroske,Cameron;Muci,John;Wang,Jicheng;Li,Kai;Song,Wenjia;Yan,Jin;Feng,Jinong;Liu,Qiang;Sommer,SteveS
虽然DOVAM-S(检测几乎所有突变-SSCP)实际上可以检测所有突变，而且比直接测序成本更低，但该技术目前需要使用放射性。F-DOVAM-S(荧光DOVAM-S)是为了用荧光取代同位素标记，并通过染料颜色复用来增加产量。作为原理的证明，通过对88例血友病B(HB)患者和7例野生型对照的凝血因子IX(FIX)基因突变的盲法分析，对两种多温平板凝胶电泳条件进行了评价。仅用两个条件，就确定了F-DOVAM-S的检测灵敏度为97%。预计当采用三到四个优化条件时，F-DOVAM-S将检测到所有突变。用三种不同的荧光染料(6FAM、VIC和NED)对三个患者样本进行每孔多重分析，表明每条凝胶通道可以分析高达44kb的二倍体彩色编码扩增产物。这一数值相当于每96孔凝胶分析的DNA吞吐量约为4Mb，约为传统放射性标记DOVAM-S的三倍。通过快速捕获荧光信号并分析所得到的多色色谱图，进一步提高了吞吐量。考虑到这些数据，F-DOVAM-S有可能成为一种特别强大的临床诊断技术，因为它允许在24小时内对多名患者进行突变分析。
Although DOVAM-S (detection of virtually all mutations–SSCP) in effect detects all mutations and is less costly than direct sequencing, the technique currently requires the use of radioactivity. F-DOVAM-S (fluorescent DOVAM-S) was developed to replace the isotopic label with fluorescence and to increase throughput via dye color multiplexing. As proof of principle, two multitemperature slab gel electrophoresis conditions were evaluated through the blinded analysis of mutations in the factor IX (FIX) genes of 88 hemophilia B (HB) patients and 7 wild-type controls. Using only two conditions, it was determined that F-DOVAM-S had a detection sensitivity of 97%. It is anticipated that when three or four optimized conditions are employed, F-DOVAM-S will detect all mutations. Three patient samples were multiplexed per well using three different fluorescent dyes (6FAM, VIC, and NED), demonstrating that it is possible to analyze up to 44 kb of diploid, color-coded amplification product per gel lane. This value corresponds to a throughput of approximately 4 Mb of DNA analyzed per 96-well gel, which is approximately triple that of conventional radiolabeled DOVAM-S. Throughput is further enhanced by the rapidity at which the fluorescent signal can be captured and the resultant multicolor chromatograms analyzed. Given these data, F-DOVAM-S has the potential to be a particularly powerful technology for clinical diagnosis because it allows the mutation analysis of multiple patients to be performed within 24h.