Development of Clinically Useful Single Copy FISH Probes

临床有用的单拷贝 FISH 探针的开发

• 批准号: 6879776
• 负责人: PETER K ROGAN
• 金额: $ 10万
• 依托单位: PHYLOGENETIX LABORATORIES, INC.
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-03-01 至 2008-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6879776
• 关键词: Internet; cytogenetics; fluorescent dye /probe; fluorescent in situ hybridization; genome; ionophores; nucleic acid sequence; technology /technique development

DESCRIPTION (provided by applicant): Flourescence in situ hybridization (FISH) is a diagnostic test that distinguishes chromosomal abnormalities not evident by routine chromosome banding. By tagging and hybridizing defined DNA sequences with fluorochromes, abnormalities can be detected for specific chromosomal loci at much higher resolution (within individual chromosome bands). Commercially available DNA probes, which detect the most common abnormalities, bind to chromosomal targets of 40-1000 kb in length, making them useful for detection of large molecular chromosomal rearrangements. To detect much smaller rearrangements, we have synthesized small single copy (sc) FISH probes designed by genomic sequence analysis (1.5 to 8 kilobases) (Rogan et al, 2001). scFISH permits examination of the chromosome at the resolution of the Southern blot (~2kb). This increased genomic resolution has significant clinical, research and commercial implications, since abnormalities indistinguishable with commercial probes can be classified into distinct categorieswith scFISH probes. However, as a result of their shorter length and reduced chromosomal targets, the fluorescent hybridization signals are, in some instances, smaller than those seen using commercially-available probes. We aim (1) to develop a web-based application to simplify the selection of scFISH probes throughout the genome and (2) to increase the hybridization signal intensity of small scFISH probes by testing novel DNA labeling/detection technologies and benchmarking them against our currently validated protocol of indirect labeling and detection with fluorophore-conjugated antibodies. We will evaluate two approaches for increasing hybridization signal intensities of hybridized probes with reagents containing either high multiplicity fluorescent adducts (DNA dendrimers) or high quantum yield semicrystalline nanoparticles. The long range goal is to make sc probes as easy to select and visualize as conventional FISH probes so that they can be introduced into clinical cytogenetic laboratories.

描述（由申请方提供）：荧光原位杂交（FISH）是一种诊断试验，用于区分常规染色体显带不明显的染色体异常。通过用荧光染料标记和杂交确定的DNA序列，可以以高得多的分辨率（在单个染色体带内）检测特定染色体位点的异常。可检测最常见异常的市售DNA探针与长度为40-1000 kb的染色体靶标结合，使其可用于检测大分子染色体重排。为了检测小得多的重排，我们合成了通过基因组序列分析设计的小单拷贝（sc）FISH探针（1.5至8个酶）（Rogan等，2001）。scFISH允许在Southern印迹的分辨率（~2kb）下检查染色体。这种增加的基因组分辨率具有重要的临床，研究和商业意义，因为与商业探针无法区分的异常可以用scFISH探针分为不同的类别。然而，由于其较短的长度和减少的染色体靶标，在某些情况下，荧光杂交信号小于使用市售探针所见的那些。我们的目标是（1）开发一个基于网络的应用程序，以简化整个基因组的scFISH探针的选择和（2）通过测试新的DNA标记/检测技术，并将其与我们目前验证的间接标记和检测方案进行基准测试，以增加小scFISH探针的杂交信号强度。我们将评估两种方法，用于增加杂交探针的杂交信号强度与试剂含有高多重性荧光加合物（DNA树枝状聚合物）或高量子产率半结晶纳米粒子。长期目标是使sc探针像常规FISH探针一样容易选择和可视化，以便它们可以被引入临床细胞遗传学实验室。