Nutiu Razvan, Li Yingfu
Department of Biochemistry, McMaster University, Health Sciences Center, 1200 Main Street West, Hamilton, ON L8N 3Z5, Canada.
Nucleic Acids Res. 2002 Sep 15;30(18):e94. doi: 10.1093/nar/gnf093.
Molecular beacons (MBs) are hairpin-like fluorescent DNA probes that have single-mismatch detection capability. Although they are extremely useful for many solution-based nucleic acid detections, MBs are expensive probes for applications that require the use of a large number of different DNA probes due to the high cost and tedious procedures associated with probe synthesis and purification. In addition, since both ends of MB probes are covalently modified with chromophores, they do not offer the flexibility for fluorophore change and the capability for surface immobilization through free DNA ends. In this report, we describe an alternative form of MB, denoted tripartite molecular beacon (TMB), that may help overcome these problems. A TMB uses an unmodified oligodeoxyribonucleotide that forms a MB-like structure with two universal single-stranded arms to bring on a universal pair of oligodeoxyribonucleotides modified separately with a fluorophore and a quencher. We found that TMBs are as effective as standard MBs in signaling the presence of matching nucleic acid targets and in precisely discriminating targets that differ by a single nucleotide. TMBs have the necessary flexibility that may make MBs more affordable for various nucleic acid detection applications.
分子信标（MBs）是具有单碱基错配检测能力的发夹状荧光DNA探针。尽管它们在许多基于溶液的核酸检测中极为有用，但由于与探针合成和纯化相关的高成本及繁琐程序，对于需要使用大量不同DNA探针的应用而言，MBs是昂贵的探针。此外，由于MB探针的两端都用发色团进行了共价修饰，它们在荧光团更换方面缺乏灵活性，也没有通过游离DNA末端进行表面固定的能力。在本报告中，我们描述了一种MB的替代形式，称为三方分子信标（TMB），它可能有助于克服这些问题。TMB使用未修饰的寡脱氧核糖核苷酸，该寡脱氧核糖核苷酸与两个通用单链臂形成类似MB的结构，以引入分别用荧光团和猝灭剂修饰的一对通用寡脱氧核糖核苷酸。我们发现，TMB在指示匹配核酸靶标的存在以及精确区分相差单个核苷酸的靶标方面与标准MBs一样有效。TMB具有必要的灵活性，这可能使MBs在各种核酸检测应用中更具成本效益。
