SBIR Phase II: High-Throughput Multi-Analyte Chromatin Immunoprecipitation (ChIP) Assay Development

这个小型企业创新研究(SBIR)第二阶段项目建议开发一种高通量、多分析物的染色质免疫沉淀(CHIP)分析方法。ChIP是生命科学和生物医学研究人员广泛使用的一种技术，他们试图了解组蛋白翻译后修饰的表观遗传机制如何影响通过染色质-蛋白质相互作用调节的各种生物功能。分析的范围从单基因(使用聚合酶链式反应)到全基因组(下一代测序(NGS))。正如设想的那样，这项拟议的技术将把冗长而繁琐的多天芯片转变为高通量兼容的单日实验。抗体与包含NGS平台兼容标签和“条形码”序列的寡核苷酸相连，用于同时分析多个DNA-蛋白质相互作用。将寡核苷酸插入染色质抗体结合染色质片段两侧的位置，将使(通过聚合酶链式反应或NGS)直接检测与感兴趣的蛋白质(S)相关的DNA片段成为可能。第二阶段的工作将集中于开发强大和可重复的方法，用于抗体结合和技术验证，使用一组针对不同类别染色质相关蛋白的抗体，包括组蛋白和非组蛋白靶标。通过新方法产生的DNA文库与传统芯片的DNA文库将首先通过定量PCR进行基因特异性分析，然后通过NGS进行全基因组分析。如果该项目成功，该项目的更广泛的影响/商业潜力将是开发一种高通量的多分析物芯片分析方法，将在生命科学和生物医学研究领域产生重大的科学和商业影响。在推出后不到五年的时间里，它预计将取代传统的当前芯片方法，后者占表观遗传研究工具市场的20%-25%，估计2010年市场价值为1.75亿-2.45亿美元。生命科学研究工具市场目前估计为420亿美元，表观遗传部门享受着高增长，这是由于研究人员转向购买商业表观遗传产品而不是内部制造的产品，以及NGS的进步，这加快了全基因组表观遗传分析的速度。这种方法的开发将通过消除技术障碍和显著减少与传统芯片相关的样本量要求(包括进行单细胞分析的可能性)，向几乎所有研究人员开放表观遗传学分析。这项技术的发展将促进其他新技术的创造，如用于高通量筛选的同质芯片、多分析芯片，并为环境、营养和毒理学学科在基因组范围内研究任何真核生物的表观遗传学图谱打开大门。

This Small Business Innovation Research (SBIR) Phase II project proposes to develop a high-throughput, multi-analyte chromatin immunoprecipitation (ChIP) assay. ChIP is a widely used technique among life science and biomedical researchers seeking to understand how the epigenetic mechanism of histone post-translational modifications impacts the varied biological functions that are regulated through chromatin-protein interactions. Analysis ranges from single gene (using PCR) through to genome-wide (next generation sequencing (NGS)). As envisioned, the proposed technology will transform the lengthy and cumbersome multi-day ChIP into a high-throughput compatible single-day experiment. An antibody is linked to oligonucleotides containing NGS platform-compatible tags and "bar-code" sequences for simultaneous analysis of multiple DNA-protein interactions. The insertion of the oligonucleotides into chromatin at sites flanking antibody-bound chromatin fragments will enable direct detection (by PCR or NGS) of DNA fragments associated with the protein(s) of interest. Phase II efforts will focus on developing robust and reproducible methods for both antibody conjugation and technology validation with a panel of antibodies specific for varying classes of chromatin associating proteins, including histone and non-histone targets. DNA libraries produced by the novel method and that of traditional ChIP will be compared first by quantitative PCR for gene-specific analysis and subsequently genome-wide, by NGS.The broader impact/commercial potential of this project, if successful, will be the development of a high throughput multi-analyte ChIP assay that will have significant impact scientifically and commercially in the life sciences and biomedical research arenas. In less than five years post-launch, it is projected to replace traditional current ChIP methods, which represents 20-25% of the epigenetic research tools market estimated at $175-245M in 2010. The life sciences research tool market is currently estimated at $42 billion, with the epigenetic sector enjoying high growth fueled by a shift of researchers to purchase commercial epigenetic products rather than in-house made, and by advances in NGS, which has accelerated genome-wide epigenetic analyses. The development of this method will open epigenetic analysis to virtually all researchers by eliminating technical barriers and by significantly reducing sample size requirements associated with traditional ChIP, including a potential for single cell analysis. Development of this technology will spur the creation of additional novel technologies such as homogeneous ChIP for high-throughput screening, multi-analyte ChIP, and open the door for environmental, nutrition, and toxicology disciplines to study the epigenetic profiles of any eukaryotic organism a genome wide scale.