MULTIPLEX CHEMILUMINESCENT DNA SEQUENCING

多重化学发光 DNA 测序

• 批准号: 2645475
• 负责人: Hashem Akhavan-Tafti
• 金额: $ 7.5万
• 依托单位: LUMIGEN, INC.
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-05-01 至 1998-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2645475
• 关键词: DNA; gel electrophoresis; luminescence; method development; nucleic acid probes; nucleic acid sequence

DESCRIPTION (Adapted from investigator's abstract): This project proposes to demonstrate the feasibility of a novel multiplexed chemiluminescent system for manual DNA sequencing or detection of double-stranded DNA that can potentially replace the use of radioprobes in small scale sequencing applications. the multiplexed detection is achieved using two enzyme labels and two chemiluminescent reagents to allow rapid, consecutive detection of two different enzyme-labeled probes on the same lane of the blotting membrane. This will allow sequence analysis to be carried out in two lanes, instead of requiring four as is the case in radiolabeling. Specially, the analysis is carried out by using primers that have specific haptens labeled onto them. After electrophoresis is carried out, the electrophoresis bands are transferred to a nylon membrane and exposed to a binding agent to the hapten that contains either HRP or AP. After rinsing, reagents are added to develop the chemiluminescent reaction for HRP using the chromophobe, Lumigen PS-3 (developed in house). After the image of the gel is acquired, this reaction is quenched by adding excess hydrogen peroxide and high pH. Then, the reagents for the AP reaction are added, including the chromophobe for this chemiluminescent reaction, Lumigen PPD. This approach allows either detection of two different termination products per lane or two different templates per lane. In essence, the approach allows doubling the throughput of manual sequencing. PROPOSED COMMERCIAL APPLICATION: Not available.

描述（改编自研究者摘要）：本项目 建议证明一种新的多路复用的可行性， 用于手动DNA测序或检测的荧光系统 双链DNA有可能取代放射性探针的使用 在小规模测序应用中。 所述多路检测 使用两种酶标记和两种荧光试剂实现， 允许快速、连续检测两种不同的酶标记 探针在印迹膜的同一泳道上。 这将允许 在两个泳道中进行序列分析，而不是要求 四是放射性标记的情况。 特别是，分析认为， 通过使用具有标记在其上的特异性半抗原的引物 他们 进行电泳后， 转移到尼龙膜上并暴露于粘合剂， 含有HRP或AP的半抗原。 冲洗后， 加入，以使用 Lumigen PS-3（内部开发）。 在图像之后， 获得凝胶，通过加入过量的氢气淬灭该反应 然后，加入AP反应的试剂， 包括这种荧光反应的发色体Lumigen PPD 这种方法允许检测两种不同的终止 每个通道的产品或每个通道两个不同的模板。 本质上 该方法允许使手动测序的通量加倍。 拟议的商业应用：不可用。