Ligase chain reaction coupled with rolling circle amplification for high sensitivity detection of single nucleotide polymorphisms
Ligase chain reaction coupled with rolling circle amplification for high sensitivity detection of single nucleotide polymorphisms
复制标题
连接酶链式反应与滚环扩增相结合，用于高灵敏度检测单核苷酸多态性
DOI：
10.1039/c3an36920j
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发表时间：
2013-01-01
期刊：
影响因子：
4.2
通讯作者：
Li, Zhengping
中科院分区：
文献类型：
--
作者：
Cheng, Yongqiang;Zhao, Jingjing;Li, Zhengping
我们提出了一种高灵敏度和同质检测单核苷酸多态性（SNPs）的连接酶链反应（LCR）结合滚环扩增（RCA）。LCR探针包括一对探针和一个挂锁探针（PLP）。在LCR中，由X和Y组成的一对探针在热变性后与靶DNA的上链完全杂交。然后它们通过热稳定连接酶连接以形成XY的连接产物。同时，PLP与靶DNA的下链杂交并连接以形成环状PLP（cPLP）。在变性、退火和连接的重复循环后，靶DNA以指数方式扩增以产生大量XY和cPLP。随后，RCA由作为模板的cPLP和作为引物的XY触发，产生大量长链DNA产物，其通过与荧光染料SYBR绿色I以均匀方式结合来检测。该方法简单，并且避免了需要用标记探针检测LCR产物和复杂的分离步骤。该检测试剂盒的灵敏度和特异性足以检测1 fM靶DNA分子。有可能精确地确定低至1.0%的等位基因频率。LCR与RCA联用技术扩展了LCR和RCA的应用范围，为SNPs检测以及核酸分析、免疫分析和分子诊断提供了新的策略。
We present a highly sensitive and homogeneous assay for the detection of single nucleotide polymorphisms (SNPs) by ligase chain reaction (LCR) coupled with rolling circle amplification (RCA). The LCR probes include one pair of probes and a padlock probe (PLP). In the LCR, one pair of probes composed of X and Y, perfectly hybridize with the upper strand of the target DNA after thermal denaturation. They are then ligated by the thermostable ligase to form the ligation product of XY. At the same time, the PLP hybridizes with the lower strand of the target DNA and are ligated to form the circular PLP (cPLP). After repeated cycles of denaturation, annealing, and ligation, the target DNA is amplified exponentially to generate a large number of XY and cPLPs. Subsequently, RCA is triggered by the cPLP as a template and XY as a primer, producing large numbers of long strand DNA products, which are detected by binding with the fluorescent dye, SYBR Green I, in a homogeneous manner. This method is simple, and avoids the need for detection of the LCR products with labeled probes and complex separation steps. The assay is sensitive and specific enough to detect a 1 fM target DNA molecule. It is possible to accurately determine the allele frequency as low as 1.0%. The LCR coupled with RCA assay extends the application of the LCR and RCA, and provides a new strategy for detecting SNPs as well as nucleic acid analysis, immunoassay, and molecular diagnosis.