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

这个小型企业创新研究（SBIR）第二阶段项目提出开发一种高通量，多分析物染色质免疫沉淀（ChIP）测定。ChIP是生命科学和生物医学研究人员广泛使用的技术，旨在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。分析范围从单个基因（使用PCR）到全基因组（下一代测序（NGS））。正如所设想的那样，所提出的技术将把冗长繁琐的多日ChIP转化为高通量兼容的单日实验。将抗体连接到含有NGS平台相容性标签和“条形码”序列的寡核苷酸上，用于同时分析多种DNA-蛋白质相互作用。寡核苷酸在抗体结合的染色质片段侧翼的位点处插入染色质将使得能够直接检测（通过PCR或NGS）与感兴趣的蛋白质相关的DNA片段。第二阶段的工作将集中在开发强大的和可重复的方法，用于抗体偶联和技术验证，其中一组抗体对不同类别的染色质相关蛋白具有特异性，包括组蛋白和非组蛋白靶标。 通过新方法和传统的ChIP产生的DNA文库将首先通过定量PCR进行基因特异性分析，随后通过NGS进行全基因组分析。如果成功，该项目的更广泛影响/商业潜力将是开发高通量多分析物ChIP检测，这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ChIP方法，这代表了2010年估计为1.75 - 2.45亿美元的表观遗传研究工具市场的20-25%。 生命科学研究工具市场目前估计为420亿美元，表观遗传领域的高增长得益于研究人员转向购买商业表观遗传产品而不是内部制造，以及NGS的进步，这加速了全基因组表观遗传分析。 这种方法的开发将通过消除技术障碍和显著减少与传统ChIP相关的样本量要求（包括单细胞分析的潜力），向几乎所有研究人员开放表观遗传分析。这项技术的发展将刺激创造更多的新技术，如用于高通量筛选的均质ChIP，多分析物ChIP，并为环境，营养和毒理学学科打开大门，以研究任何真核生物的表观遗传学概况。

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.