Walker G T, Linn C P
Becton Dickinson Research Center, Research Triangle Park, NC 27709-2016, USA.
Clin Chem. 1996 Oct;42(10):1604-8.
Strand displacement amplification (SDA) is an isothermal, in vitro method for diagnostics that amplifies a target DNA sequence by using a restriction enzyme and DNA polymerase. We have combined a new thermophilic form of SDA that involves restriction enzyme BsoBI and polymerase exo-Bca with fluorescence polarization for detection of Mycobacterium tuberculosis DNA by using the IS6110 insertion element as the target sequence. A 5'-fluorescein-labeled oligodeoxynucleotide detector probe hybridizes to the amplified product as it rises in concentration during SDA, and the single- to double-stranded conversion is monitored through an increase in fluorescence polarization. The associated change in polarization upon amplification of the target sequence is enhanced by specific polymerase binding to the double-stranded detector probe. Fewer than 10 M. tuberculosis genomes can be amplified and detected with an extremely simple protocol that takes only 20 min and uses relatively simple instrumentation and reagents, all of which can be purchased off-the-shelf.
链置换扩增（SDA）是一种用于诊断的等温体外方法，它通过使用限制性内切酶和DNA聚合酶来扩增目标DNA序列。我们将一种新的嗜热形式的SDA（涉及限制性内切酶BsoBI和外切酶Bca聚合酶）与荧光偏振相结合，以IS6110插入元件作为目标序列来检测结核分枝杆菌DNA。一个5'-荧光素标记的寡脱氧核苷酸检测探针在SDA过程中随着扩增产物浓度的升高与之杂交，并且通过荧光偏振的增加来监测单链到双链的转变。目标序列扩增时偏振的相关变化通过特异性聚合酶与双链检测探针的结合而增强。通过一个极其简单的方案，仅需20分钟，使用相对简单的仪器和试剂（所有这些都可以现货购买），就可以扩增并检测到少于10个结核分枝杆菌基因组。
