Cross-Hybridization in Multiplex Hybridization Reaction

描述(由申请人提供)：许多核酸单链的多重杂交导致许多序列特异的单独的双链复合体的形成，这是许多基于核酸的诊断分析的中心过程。目前，这种检测方法被用于分析基因表达、检测基因变异和突变、评估病毒载量对治疗剂的反应以及检测感染性病原体。理想情况下，多重反应中的每一条链都意味着只与其完全匹配和互补的单链形成双链。然而，在现实中，根据存在的特定序列，许多驻留的单链也可以与其完全匹配的补体链以外的链进行退火，从而产生交叉杂交，即x-Hyb。X-Hyb是多重杂交反应的主要敌人，导致假阳性信号，降低准确度和灵敏度。尽管x-Hyb很容易被所有多重杂交分析的实践者承认为一个潜在的问题，但对x-Hyb的分子相互作用尚不清楚。除了重新设计探针和引物组并再次尝试它们(很大程度上是一种反复试验的经验方法)，如果给定的探针和/或引物组未能按设计的方式发挥作用[由于x-Hyb]，几乎没有什么办法。这个第二阶段项目的目标是定量定义x-Hyb的序列依赖特征，并开发一个在单个序列水平上诊断和预测x-Hyb的可靠分析框架。第一个具体目标涉及评估作为序列、溶液和微阵列的函数的串联错配的热力学参数，然后将这些信息纳入用于x-Hyb分析的软件工具集中。第二个具体目标是应用所获得的能力，开发基于RT-PCR和微阵列的分析方法，以检测Creld1基因的一个可替代剪接变体，即(外显子9b)异构体，该异构体的存在已被认为与各种癌症有关。

DESCRIPTION (provided by applicant): Multiplex hybridization of a number of nucleic acid single strands resulting in the formation of many sequence specific, individual duplex complexes, is the central process in a number of nucleic acid based diagnostic assays. Such assays are currently employed to analyze gene expression, detect genetic variations and mutations, assess viral loads in response to therapeutic agents and detect infectious pathogenic agents. Ideally, each strand present in a multiplex reaction is meant to form a duplex with only its perfectly matched and complementary single strand. In reality however, depending on the particular sequences present, many of the resident single strands can also anneal with strands other than their perfectly matched complement strand, resulting in cross-hybridization, x-hyb. X-hyb is a major nemesis of multiplex hybridization reactions causing false positive signals, lowered accuracy and sensitivity. Although x-hyb is readily acknowledged as a potential problem by all practicitioners of multiplex hybridization assay, the molecular interactions responsible for x-hyb are not understood. Other than redesigning the probes and primers and trying them again (very much a trial and error, empirical approach) there is little recourse should a given probe and/or primer set fail to function as designed [due to x-hyb]. The goal of this Phase II project is to quantitatively define sequence dependent features of x-hyb and develop a robust analytical framework for diagnosing and predicting x-hyb on the level of individual sequences. The first specific aim involves evaluation of thermodynamic parameters for tandem mismatches as a function of sequence, in solution and on microarrays, then incorporating this information in a software toolset for x-hyb analysis. The second specific aim is to apply the capabilities gained to develop RT-PCR and microarray based assays for detection of an alternatly spliced variant of the Creld1 gene, the (exon 9b) isoform, whose presence has been has been linked to various cancers.