Walker G T, Linn C P, Nadeau J G
Becton Dickinson Research Center, Research Triangle Park, NC 27709-2016, USA.
Nucleic Acids Res. 1996 Jan 15;24(2):348-53. doi: 10.1093/nar/24.2.348.
Strand displacement amplification (9SDA) is an isothermal in vitro method of amplifying a DNA sequence prior to its detection. We have combined SDA with fluorescence polarization detection. A 5'-fluorescein-labelled oligodeoxynucleotide detector probe hybridizes to the amplification product that rises in concentration during SDA and the single- to double strand conversion is monitored through an increase in fluorescence polarization. Detection sensitivity can be enhanced by using a detector probe containing an EcoRI recognition sequence at its 5'-end that is not homologous to the target sequence. During SDA the probe is converted to a fully double-stranded form that specifically binds a genetically modified form of the endonuclease EcoRI which lacks cleavage activity but retains binding specificity. We have applied this SDA detection system to a target sequence specific for Mycobacterium tuberculosis.
链置换扩增（SDA）是一种在体外等温条件下，在检测DNA序列之前对其进行扩增的方法。我们将SDA与荧光偏振检测相结合。一个5'-荧光素标记的寡脱氧核苷酸检测探针与在SDA过程中浓度升高的扩增产物杂交，通过荧光偏振的增加来监测单链到双链的转变。通过使用在其5'-末端含有一个与靶序列不同源的EcoRI识别序列的检测探针，可以提高检测灵敏度。在SDA过程中，该探针被转化为完全双链形式，它能特异性结合一种经过基因改造的内切酶EcoRI，这种EcoRI缺乏切割活性但保留结合特异性。我们已将这种SDA检测系统应用于结核分枝杆菌特异性的靶序列。
