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Studies in detection limits and selectivity of probes used for the assessment of histone acetylation at specific loci.

机译：研究用于检测特定位点组蛋白乙酰化的探针的检出限和选择性。

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摘要

Highly sensitive and selective probes for screening historic acetylation at particular loci are necessary. This work describes the design of selective DNA probes for the identification of specific chromosomal regions and highlights the importance of signal amplification for probes of less than 2 kilobases (kb), and the effect that particle size used for the amplification may have on its ability to bind to the target. Single copy FISH probes were designed for the identification of target regions, and generated by PCR amplification. Probe characterization was done by nick translation labeling with biotin or digoxigenin and detection with avidin or anti-digoxigenin. The probes proved to be selective, with detection efficiencies between 50 and 90%. Different labeling approaches were evaluated for the use of 3D DNA structures (dendrimers) to amplify the scFISH probe fluorescent signal. Low signal amplification of a 2.4 kb probe with anti-biotin dendrimers suggested that steric hindrance might keep dendrimers from binding to target probes. Qualitative studies of samples immobilized in polyacrylamide by near confocal microscopy demonstrated that after incubation with dendrimers there were biotins still available for binding. Furthermore, detection of higher number of quantum dots (15 nm in diameter) bound to probe than dendrimers (90 nm diameter) suggested that particle size will have to be considered when designing a method for signal amplification.For the detection of histone acetylation, antibodies against H4-Ac16 and H4-nonAc were generated by conjugation of the target peptide to PPD. After affinity purification, the cross-reactivity of these antibodies to the non-antigenic peptide was &sim8%. Immunofluorescence experiments with commercial and in-house antibodies on chromatin extended fibers agreed with the "zip" model of acetylation for H4, where the order of acetylation is from lysine 16 to lysine 5. It was infered from theoretical calculations and previous reports that the study of acetylation changes at regions of &sim2.4 kb would require detection of modifications in 28 histone molecules, indicating the need for signal amplification for the determination.
机译：必须使用高灵敏度和选择性的探针来筛选特定位点的历史乙酰化。这项工作描述了用于鉴定特定染色体区域的选择性DNA探针的设计，并着重强调了信号放大对于小于2 KB的探针的重要性，以及用于扩增的粒径可能对其信号强度的影响。绑定到目标。单拷贝FISH探针设计用于鉴定目标区域，并通过PCR扩增产生。通过用生物素或洋地黄毒苷进行切口平移标记并用抗生物素蛋白或抗地高辛原进行检测来进行探针鉴定。探针被证明具有选择性，检测效率在50％至90％之间。针对使用3D DNA结构（树状聚合物）来扩增scFISH探针荧光信号的方法，评估了不同的标记方法。用抗生物素树状聚合物对2.4 kb探针进行的低信号扩增表明，空间位阻可能会使树状聚合物无法与靶探针结合。通过近共聚焦显微镜对固定在聚丙烯酰胺中的样品进行定性研究表明，与树状大分子一起孵育后，仍有生物素可用于结合。此外，与树状聚合物（直径90 nm的直径）相比，与探针结合的量子点（直径15 nm）的数量更多，这表明在设计信号放大方法时必须考虑粒径。对于组蛋白乙酰化的检测，抗体通过将靶肽与PPD缀合产生针对H4-Ac16和H4-nonAc的抗。亲和纯化后，这些抗体与非抗原肽的交叉反应性为＆sim8％。在染色质延伸纤维上使用商业抗体和内部抗体进行的免疫荧光实验与H4乙酰化的“拉链”模型相吻合，其中乙酰化的顺序是从赖氨酸16到赖氨酸5。这是从理论计算和以前的报道中推断的如果要在＆sim2.4 kb的区域进行乙酰化检测，则需要检测28个组蛋白分子中的修饰，这表明需要进行信号放大。

著录项

  • 作者

    Mora, Johanna.;

  • 作者单位

    University of Kansas.;

  • 授予单位 University of Kansas.;
  • 学科 Chemistry Analytical.
  • 学位 Ph.D.
  • 年度 2004
  • 页码 184 p.
  • 总页数 184
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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