Multiplexed DNA sequencing and diagnostics by hybridization with enriched stable isotope labels.
Multiplexed DNA sequencing and diagnostics by hybridization with enriched stable isotope labels.
复制标题
通过与富集的稳定同位素标记杂交进行多重 DNA 测序和诊断。
DOI：
10.1021/ac961206e
复制
发表时间：
1997
影响因子：
7.4
通讯作者：
Jacobson,KB
中科院分区：
文献类型：
--
作者：
Arlinghaus,HF;Kwoka,MN;Guo,XQ;Jacobson,KB
一种新的 DNA 诊断和测序系统已经开发出来，该系统使用飞行时间共振电离质谱 (TOF-RIMS) 提供一种在 1 型杂交测序 (SBH) 中分析稳定同位素标记寡核苷酸的快速方法。对于形式 1，DNA 固定在尼龙膜上，富集同位素标记的单个寡核苷酸探针在杂交过程中可以自由寻找互补 DNA。这种新方法的主要优点是多种寡核苷酸可以用不同的富集同位素标记，并且可以同时与基因传感器基质杂交。然后可以使用 TOF-RIMS 同时检测探针，具有高选择性、灵敏度和效率。通过使用同位素富集的锡标记，在与 DNA 基质的单次杂交中可以检查多达 10 个标记的寡核苷酸。如果使用稀土同位素，则可以获得更多数量的标记。在本研究中，制备了含有三种不同DNA的基质，并在多种条件下同时与两种不同的探针杂交。结果表明，固定在尼龙表面上的 DNA 可以与用不同富集锡同位素标记的探针特异性杂交。在多重样品中，互补位点和非互补位点之间的区分度优于 100。这种新的 SBH 方法采用稳定同位素标记来定位目标 DNA，并使用 TOF-RIMS 来检测标记，将是一种非常通用且广泛的多重方法。
A new DNA diagnostic and sequencing system has been developed that uses time-of-flight resonance ionization mass spectrometry (TOF-RIMS) to provide a rapid method of analyzing stable isotope-labeled oligonucleotides in form 1 sequencing by hybridization (SBH). With form 1, the DNA is immobilized on a nylon membrane and enriched isotope-labeled individual oligonucleotide probes are free to seek out complementary DNAs during hybridization. The major advantage of this new approach is that multiple oligonucleotides can be labeled with different enriched isotopes and can all be simultaneously hybridized to the genosensor matrix. The probes can then be simultaneously detected with TOF-RIMS with high selectivity, sensitivity, and efficiency. By using isotopically enriched tin labels, up to 10 labeled oligonucleotides could be examined in a single hybridization to the DNA matrix. Greater numbers of labels are available if rare earth isotopes are employed. In the present study, matrices containing three different DNAs were prepared and simultaneously hybridized with two different probes under a variety of conditions. The results show that DNAs, immobilized on nylon surfaces, can be specifically hybridized to probes labeled with different enriched tin isotopes. Discrimination between complementary and noncomplementary sites of better than 100 was obtained in multiplexed samples. This new SBH method, which employs stable isotopic labels to locate target DNAs and TOF-RIMS to detect the labels, will be a very versatile and extensive multiplexing method.