Harvey John J, Brant Steven R, Knutson Jay R, Han Myun K
Excimus Biotech, Inc., 8510 Corridor Road, Savage, MD 20763, USA.
J Clin Lab Anal. 2008;22(3):192-203. doi: 10.1002/jcla.20240.
CataCleave probes are catalytically cleavable fluorescence probes having a chimeric deoxyribonucleic acid (DNA)-ribonucleic acid (RNA)-DNA structure that can be used for real-time detection of single nucleotide polymorphisms (SNPs), insertions, and deletions. Fluorescent donor emission is normally quenched by Förster resonance energy transfer (FRET). Upon binding to the target, if the RNA/DNA hybrid is correctly base-paired, ribonuclease (RNase) H will cleave the RNA moiety and the probe fragments will dissociate. FRET is lost and the donor fluorescence signal is recovered. A single-base mismatch within the hybrid region causes probe cleavage to be significantly reduced. We designed CataCleave probes to detect SNPs located in the insulin-like growth factor 2 (IGF-2) gene and at position 702 within the NOD2/CARD15 gene. Probes were also designed to detect a six-basepair deletion in the amelogenin gene and a partially methylated target DNA. Discrimination between wild-type and SNP is demonstrated for both genes in homogeneous reactions under isothermal and temperature cycling conditions. These probes were also able to identify a multibase deletion and methylated DNA. Cleavage rates were proportional to target concentration. Probe length and position of fluorescent labels may also be modified for use in multiplexing high-throughput SNP assays. This represents a novel method for the detection of SNPs.
催化切割探针是具有嵌合脱氧核糖核酸（DNA）-核糖核酸（RNA）-DNA结构的可催化切割的荧光探针，可用于实时检测单核苷酸多态性（SNP）、插入和缺失。荧光供体发射通常通过福斯特共振能量转移（FRET）被淬灭。与靶标结合后，如果RNA/DNA杂交体碱基正确配对，核糖核酸酶（RNase）H将切割RNA部分，探针片段将解离。FRET消失，供体荧光信号恢复。杂交区域内的单碱基错配会导致探针切割显著减少。我们设计了催化切割探针来检测位于胰岛素样生长因子2（IGF-2）基因以及NOD2/CARD15基因第702位的SNP。还设计了探针来检测牙釉蛋白基因中的六碱基对缺失以及部分甲基化的靶标DNA。在等温及温度循环条件下的均相反应中，对这两个基因均证明了野生型和SNP之间的区分。这些探针还能够识别多碱基缺失和甲基化DNA。切割速率与靶标浓度成正比。探针长度和荧光标记位置也可进行修饰，以用于多重高通量SNP检测。这代表了一种检测SNP的新方法。
