Identification of Single-Nucleotide Polymorphisms by the Oligonucleotide Ligation Reaction: A DNA Biosensor for Simultaneous Visual Detection of Both Alleles
Identification of Single-Nucleotide Polymorphisms by the Oligonucleotide Ligation Reaction: A DNA Biosensor for Simultaneous Visual Detection of Both Alleles
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DOI：
10.1021/ac801870x
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发表时间：
2009-01-01
影响因子：
7.4
通讯作者：
Flordellis, Christodoulos S.
中科院分区：
文献类型：
--
作者：
Toubanaki, Dimitra K.;Christopoulos, Theodore K.;Flordellis, Christodoulos S.
虽然单核苷酸多态(SNPs)可以通过与等位基因特异的寡核苷酸探针直接杂交来鉴定，但基于酶的基因分型方法提供了更高的特异性和稳健性。在酶学方法中，寡核苷酸连接反应(OLR)对等位基因的识别具有最高的特异性，因为连接需要两个杂交事件。我们报道了一种DNA生物传感器的开发，它比目前可用的检测OLR产物的方法具有显著的优势：它允许使用单个连接反应同时对两个等位基因进行视觉识别。检测在几分钟内完成，不需要任何专门的仪器。它不涉及孵化和清洗的多个周期。干试剂格式最大限度地减少了移液步骤。对合格人员的需求比目前的方法低得多。该方法的原理如下：在PCR扩增后，用一个生物素标记的普通探针和两个用地高辛和荧光素标记的半抗原标记的等位基因特异性探针进行一次OLR。对应于正常和突变等位基因的连接产物分别用生物素和地高辛或荧光素进行双重标记。这些产物被固定在生物传感器的两个测试区的抗地高辛和/或抗荧光素抗体捕获，并与抗生素功能化的金纳米颗粒记者反应。多余的纳米颗粒与固定在生物传感器控制区的生物素化白蛋白结合。基因型由两个测试区出现的特征红线指定。建议的DNA生物传感器构成了朝着看护点SNP基因分型迈出的重要一步。
Although single nucleotide polymorphisms (SNPs) can be identified by direct hybridization with allele-specific oligonucleotide probes, enzyme-based genotyping methods offer much higher specificity and robustness. Among enzymatic methods, the oligonucleotide ligation reaction (OLR) offers the highest specificity for allele discrimination because two hybridization events are required for ligation. We report the development of a DNA biosensor that offers significant advantages over currently available methods for detection of OLR products: It allows simultaneous visual discrimination of both alleles using a single ligation reaction. Detection is complete within minutes without the need for any specialized instruments. It does not involve multiple cycles of incubation and washing. The dry-reagent format minimizes the pipetting steps. The need for qualified personnel is much lower than current methods. The principle of the assay is as follows: Following PCR amplification, a single OLR is performed using a biotinylated common probe and two allele-specific probes labeled with the haptens digoxigenin and fluorescein. Ligation products corresponding to the normal and mutant allele are double-labeled with biotin and either digoxigenin or fluorescein, respectively. The products are captured by antidigoxigenin or antifluorescein antibodies, or both, that are immobilized at the two test zones of the biosensor and react with antibiotin-functionalized gold nanoparticle reporters. The excess nanoparticles bind to biotinylated albumin that is immobilized at the control zone of the biosensor. The genotype is assigned by the characteristic red lines that appear at the two test zones. The proposed DNA biosensor constitutes a significant step toward point-of-care SNP genotyping.