Grossman P D, Bloch W, Brinson E, Chang C C, Eggerding F A, Fung S, Iovannisci D M, Woo S, Winn-Deen E S
Applied Biosystems Division, Perkin Elmer Corporation, Foster City, CA 94404.
Nucleic Acids Res. 1994 Oct 25;22(21):4527-34. doi: 10.1093/nar/22.21.4527.
We describe a non-isotopic, semi-automated method for large-scale multiplex analysis of nucleic acid sequences, using the cystic fibrosis transmembrane regulator (CFTR) gene as an example. Products of a multiplex oligonucleotide ligation assay (OLA) are resolved electrophoretically from one another and from unligated probes under denaturing conditions with fluorescence detection. One ligation probe for each OLA target carries a fluorescent tag, while the other probe carries an oligomeric non-nucleotide mobility modifier. Each OLA product has a unique electrophoretic mobility determined by the ligated oligonucleotides and the mobility-modifier oligomer arbitrarily assigned (coded) to its target. The mobility range for practical mobility modifiers is much wider than the accessible range from unmodified ligated oligonucleotides of practical length. Each mobility modifier is built from phosphoramidite monomers in a stepwise manner on its associated oligonucleotide using an automated synthesizer. The resulting mobility modifiers lower the probe-target duplex Tm by less than 3 degrees C and retard probe-target annealing by less than 50%, with negligible effect on OLA yield and specificity. This method is especially useful for allelic discrimination in highly polymorphic genes such as CFTR.
我们描述了一种非同位素的半自动化方法,用于大规模多重核酸序列分析,以囊性纤维化跨膜调节因子(CFTR)基因为例。多重寡核苷酸连接分析(OLA)的产物在变性条件下通过荧光检测进行电泳分离,彼此之间以及与未连接的探针分离。每个OLA靶标的一个连接探针带有荧光标签,而另一个探针带有寡聚非核苷酸迁移率修饰剂。每个OLA产物具有独特的电泳迁移率,由连接的寡核苷酸和任意分配(编码)给其靶标的迁移率修饰剂寡聚物决定。实际迁移率修饰剂的迁移率范围比实际长度的未修饰连接寡核苷酸的可及范围宽得多。每个迁移率修饰剂使用自动合成仪在其相关寡核苷酸上以逐步方式由亚磷酰胺单体构建。所得迁移率修饰剂使探针 - 靶标双链体的Tm降低不到3摄氏度,使探针 - 靶标退火延迟不到50%,对OLA产量和特异性的影响可忽略不计。该方法对于高度多态性基因(如CFTR)中的等位基因鉴别特别有用。