Maldonado-Rodriguez R, Espinosa-Lara M, Loyola-Abitia P, Beattie W G, Beattie K L
Escuela Nacional de Ciencias Biológicas, IPN, México, DF.
Mol Biotechnol. 1999 Feb;11(1):13-25. doi: 10.1007/BF02789173.
A new strategy for analysis of point mutations using oligonucleotide array (genosensor) hybridization was investigated. In the new approach, a single-stranded target strand is preannealed with a labeled "stacking oligonucleotide," and then the partially duplex labeled target molecule is hybridized to an array of glass-tethered oligonucleotide probes, targeted to the region on the target immediately adjacent to the stacking oligomer. In this configuration, the base-stacking interactions between the "capture probe" and the contiguously stacking oligomer stabilize the binding of the target molecule to its complementary probe on the genosensor array. The temperature of hybridization can be adjusted so that the target molecule will bind to the glass-tethered probe only in the presence of the stacking oligomer, and a single mismatch at or near the terminal position ol the capture probe disrupts the stacking interactions and thereby eliminates or greatly reduces the hybridization. This stacking hybridization approach was investigated using a collection of synthetic targets, probes, and stacking oligonucleotides, which permitted identification of conditions for optimal base mismatch discrimination. The oligonucleotide probes were tethered to the glass using a simple, improved attachment chemistry in which a 3'-aminopropanol function introduced into the probe during chemical synthesis binds covalently to silanol groups on clean, underivatized glass. "Operating parameters" examined in the stacking hybridization system included length of capture probe, position, type and number of mismatches between the probe and the target, temperature of hybridization and length of washing, and the presence of terminal phosphate group in the probe, at its junction with the stacking oligomer. The results suggest that in the stacking hybridization configuration: 1. Optimal mismatch discrimination with 9-mer probes occurs at 45 degrees C, after which little or no improvement in mispair rejection occurred on lengthy continued washing at 45 degrees C. 2. At 25 degrees C optimal mismatch discrimination occurred with 7- or 8-mer probes, or with 9-mer probes containing an additional internal mismatch. 3. The presence of a phosphate group on the 5'-end of the glass-tethered probe had no general effect on mismatch discrimination, but influenced the relative stability of different mismatches in the sequence context studied. These results provide a motivation for continued development of the stacking hybridization technique for nucleic acid sequence analysis. This approach offers several advantages over the traditional allele-specific oligonucleotide hybridization technique, and is distinct from the contiguous stacking hybridization sitrategy that the Mirzabekov laboratory has introduced (Yershov et al. (1996) Proc. Natl. Acad. Sci. USA 93, 4913-4918; Parinov et al. (1996) Nucleic Acids Res. 24, 2998-3004).
研究了一种使用寡核苷酸阵列（基因传感器）杂交分析点突变的新策略。在这种新方法中，单链靶标链先与标记的“堆积寡核苷酸”预退火，然后将部分双链标记的靶标分子与一系列玻璃连接的寡核苷酸探针杂交，这些探针靶向靶标上紧邻堆积寡聚物的区域。在这种配置下，“捕获探针”与连续堆积的寡聚物之间的碱基堆积相互作用稳定了靶标分子与基因传感器阵列上其互补探针的结合。可以调整杂交温度，使得靶标分子仅在存在堆积寡聚物时才与玻璃连接的探针结合，并且捕获探针末端位置或附近的单个错配会破坏堆积相互作用，从而消除或大大降低杂交。使用一组合成靶标、探针和堆积寡核苷酸对这种堆积杂交方法进行了研究，这使得能够确定最佳碱基错配区分的条件。寡核苷酸探针通过一种简单的、改进的连接化学方法连接到玻璃上，即在化学合成过程中引入到探针中的3'-氨基丙醇官能团与清洁的、未衍生化的玻璃上的硅醇基团共价结合。在堆积杂交系统中研究的“操作参数”包括捕获探针的长度、错配的位置、探针与靶标之间错配的类型和数量、杂交温度和洗涤时间，以及探针在与堆积寡聚物连接处的末端磷酸基团的存在情况。结果表明，在堆积杂交配置中：1. 使用9聚体探针时，在45℃可实现最佳错配区分，此后在45℃长时间持续洗涤对错配排斥几乎没有改善。2. 在25℃时，7聚体或8聚体探针，或含有额外内部错配的9聚体探针可实现最佳错配区分。3. 玻璃连接探针5'-末端存在磷酸基团对错配区分没有普遍影响，但会影响所研究序列背景下不同错配的相对稳定性。这些结果为继续开发用于核酸序列分析的堆积杂交技术提供了动力。这种方法相对于传统的等位基因特异性寡核苷酸杂交技术具有几个优点，并且与米尔扎别科夫实验室引入的连续堆积杂交策略不同（耶尔绍夫等人（1996年）《美国国家科学院院刊》93卷，4913 - 4918页；帕里诺夫等人（1996年）《核酸研究》24卷，2998 - 3004页）。
