NOVEL CHEMILUMINESCENT DETECTION OF GENE REARRANGEMENTS

第一阶段的目标是开发简单、敏感和明确的 化学发光法检测染色体中并列基因 易位与许多癌症有关。我们建议化学发光 基于两个基因的同时探测和检测的检测格式 一个单一的Southern印迹中的易位。第一阶段将证明 建立一种Southern杂交方法检测小麦品种的可行性 同时检测bcl-2癌基因和JH DNA T(14；18)的免疫球蛋白重链基因片段 易位。将使用两种类型实现同时检测 化学发光格式：一种双基板，它只通过 两种不同抗体-酶结合物结合的联合作用 相互接近的靶向DNA；以及连续应用 不同的化学发光底物，其中化学发光 从第一步中的一个探头生成的数据在 第二步，当第二种酶从另一种探针发出光时。 所提出的方法可以适用于多种不同的检测 与癌症相关的基因并列。分阶段开发的方法 我将在第二阶段开发快速微量试剂盒 基因重排的检测。 拟议的商业应用：拟议的化学发光分析 应为新的检测方法和试剂盒提供基础 由染色体易位引起的各种基因排列 与特定类型的癌症有关。建议的化验结果将是 比目前的基因检测方法更简单、更快速的方法 重新排列，并将允许在一次测试中进行明确的检测。 该检测系统也可用于开发工具包进行开发 在癌症治疗过程中最小的残留疾病。

The objective of Phase I is to develop simple, sensitive and unambiguous chemiluminescent detection of juxtaposed genes in chromosomal translocations associated with many cancers. We propose chemiluminescent assay formats based on simultaneous probing and detection of both genes of a translocation in a single Southern blot. Phase I will prove the feasibility by developing a Southern hybridization assay for the simultaneous detection of the juxtaposed bcl-2 oncogene and the JH DNA segment of the immunoglobulin heavy chain locus in the t(14;18) translocations. Simultaneous detection will be achieved using two types of chemiluminescent formats: a dual substrate that emits light only by the combined action of two different antibody-enzyme conjugates bound to the target DNA in close proximity to each other; and consecutive application of separate chemiluminescent substrates where the chemiluminescence generated from one probe in the first step is completely stopped in the second step when the second enzyme initiates light from the other probe. The proposed methods can be adapted to the detection of many different juxtaposed genes associated with cancers. The methods developed in Phase I will be used to Phase II to develop kits for rapid microtiter plate assays for gene rearrangements. PROPOSED COMMERCIAL APPLICATION: The proposed chemiluminescent assays should provide the basis for a new method and kits for detection of various gene arrangements resulting from chromosomal translocations associated with specific types of cancer. The proposed assays will be simpler and more rapid than current methods of detecting gene rearrangements and will permit unambiguous detection in a single test. The detection system can also be used in developing kits for developing minimal residual disease during cancer therapies.