NOVEL CHEMILUMINESCENT DETECTION OF GENE REARRANGEMENTS

基因重排的新型化学发光检测

• 批准号: 2423585
• 负责人: Hashem Akhavan-Tafti
• 金额: $ 10万
• 依托单位: LUMIGEN, INC.
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-09-01 至 1998-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2423585
• 关键词: alkaline phosphatase; bioassay; biotechnology; cardenolides; chromosome translocation; gene rearrangement; horseradish peroxidase; immunoglobulin genes; luminescence; method development; oncogenes; polymerase chain reaction; southern blotting; tissue /cell culture

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.

第一阶段的目标是开发简单、灵敏和明确的 染色体中并列基因的化学发光检测 与许多癌症相关的易位。 我们提出了发光的 基于同时探测和检测两种基因的测定形式 一次DNA印迹就发现了易位 第一阶段将证明 通过开发Southern杂交检测的可行性， bcl-2癌基因和JH DNA的同时检测 t（14;18）中免疫球蛋白重链基因座的片段 易位 同时检测将使用两种类型 发光格式：一个双基板，发射光只由 结合至抗体的两种不同抗体-酶缀合物的联合作用 彼此紧密接近靶DNA;和连续施用 其中化学发光 在第一步骤中从一个探针产生的电流在第二步骤中完全停止。 第二步，当第二种酶引发来自另一种探针的光时。 所提出的方法可以适用于许多不同的检测 与癌症相关的并列基因 阶段中开发的方法 我公司将用于二期开发试剂盒，用于快速微量滴定板 用于基因重排的测定。 拟定商业应用：拟定荧光测定 应提供一个新的方法和试剂盒检测的基础上， 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。 拟定试验将 比目前的基因检测方法更简单、更快速， 重排，并将允许在单一测试中明确检测。 所述检测系统还可用于开发试剂盒， 在癌症治疗过程中的微小残留疾病。