State Key Laboratory of Bioelectronics, Southeast University, Nanjing, 210096, China.
Molecules. 2010 Jan 27;15(2):619-26. doi: 10.3390/molecules15020619.
In this paper we describe an isothermal rolling-circle amplification (RCA) protocol to detect gene point mutations on chips. The method is based on an allele-specific oligonucleotide circularization mediated by a special DNA ligase. The probe is circularized when perfect complementary sequences between the probe oligonucleotide and HIV cDNA gene. Mismatches around the ligation site can prevent probe circularization. The circularized probe (C-probe) can be amplified by rolling circle amplification to generate multimeric singlestranded DNA (ssDNA) under isothermal conditions. There are four sequence regions to bind respectively with fluorescent probe, RCA primer, solid probe and HIV cDNA template in the C-probe which we designed. These ssDNA products are hybridized with fluorescent probes and solid probes which are immobilized on a glass slide composing a regular microarray pattern. The fluorescence signals can be monitored by a scanner in the presence of HIV cDNA templates, whereas the probes cannot be circularized and signal of fluorescence cannot be found. The RCA array has capability of high-throughput detection of the point mutation and the single-nucleotide polymorphism (SNP).The development of C-probe-based technologies offers a promising prospect for situ detection, microarray, molecular diagnosis, single nucleotide polymorphism, and whole genome amplification.
在本文中，我们描述了一种用于在芯片上检测基因突变的等温滚环扩增（RCA）方法。该方法基于一种特殊的 DNA 连接酶介导的探针寡核苷酸环化。当探针寡核苷酸与 HIV cDNA 基因之间存在完全互补序列时，探针发生环化。在连接点周围的错配可以阻止探针环化。环化的探针（C-探针）可以通过滚环扩增进行扩增，在等温条件下产生多聚单链 DNA（ssDNA）。我们设计的 C-探针中有四个序列区域分别与荧光探针、RCA 引物、固相探针和 HIV cDNA 模板结合。这些 ssDNA 产物与固定在玻璃载玻片上的荧光探针和固相探针杂交，组成规则的微阵列图案。在存在 HIV cDNA 模板的情况下，可以通过扫描仪监测荧光信号，而探针不能环化，也无法检测到荧光信号。RCA 阵列具有高通量检测点突变和单核苷酸多态性（SNP）的能力。基于 C-探针的技术的发展为原位检测、微阵列、分子诊断、单核苷酸多态性和全基因组扩增提供了有前途的前景。
