SOFTWARE AND INSTRUMENTATION TO IDENTIFY CANCER GENES

对于癌症研究人员来说，快速对基因组区域进行重新排序或确定与癌症发展有潜在关联的大量基因的差异表达谱的能力将是非常有价值的。CGAP和人类基因组计划产生了大量的DNA序列信息，这些信息可以有效地分析，然后用当代“芯片”式的重新测序或表达阵列来表示。这严重限制了可以彻底检查的临床样本的数量。我们建议构建专用阵列，该阵列具有计算机识别和设计的可立即重构的基因序列。这个分阶段的创新奖项将回答以下问题：1)阵列设计软件、数字光学化学（DOC）芯片制造设备、MAGNA/HIC读出设备和分析/基因网络软件能否构建和加固，用于通过基因表达谱分析和基于芯片的重测序对癌症样本进行常规分析，以区分癌症和非癌症细胞及其进展？2)随着新的基因组数据的积累，DOC方法所实现的可定制阵列能否不断扩展和改进，以产生专门用于分析不同癌细胞类型的芯片？3)候选cDNA序列（CGAP）和更大的基因组区域是否可以通过计算机分析（如虚拟表达阵列计算）进行重测序，以确定癌症患者群体中信息量大到snp的总变异，从而确定新的癌基因或肿瘤抑制基因？该计划的具体目标是：1)开发用于鉴定和阐明候选癌症相关基因的生物信息学工具，包括用于设计、读取和分析基因表达和重测序阵列的软件；2)开发并完成一个数字光学化学（DOC）阵列制造单元，该单元能够在单个芯片上合成至少100,000个定制寡核苷酸阵列成员，并具有每2小时快速构建不同阵列的芯片的能力；3)通过改造/复制自主研发的高光谱成像显微镜，开发构建定制的DOC读出系统；4)整合整个软硬件系统，用于DNA微阵列芯片的设计、制造和数据分析，以及随后在与癌症研究相关的临床样本上进行测试。用于突变检测、SNP发现、等位基因分型和表达分析的集成系统的测试将进展到使用档案和前瞻性收集的活检细胞和细针抽吸细胞，这些细胞已被新的激光捕获显微解剖系统纯化。这将使我们的系统与当前的主要技术相结合，产生一种工具，应该广泛用于癌症研究的转化和临床试验。

The ability to rapidly re-sequence a genomic region or to determine the differential expression profile of a large number of genes that are potentially implicated in cancer development will be extremely valuable for the cancer researcher. There is a tremendous amount of DNA sequence information being generated by the CGAP and human genome programs, more than can be effectively analyzed and then represented in contemporary 'chip' style re- sequencing or expression arrays. This severely limits the number of clinical samples that can be thoroughly inspected. We propose to construct dedicated arrays which have immediately reconfigurable gene sequences identified and designed by computer. This phased innovation award will answer the following questions: 1) Can the array design software, Digital Optical Chemistry (DOC) chip manufacturing device, the MAGNA/HIC readout device, and analysis/gene network software be constructed and ruggedized for routine analysis of cancer samples to differentiate cancer non-cancer cells and their progression by gene expression profiling and chip based resequencing? 2) Can the customizable arrays made possible by the DOC approach be continuously expanded and improved as new genomic data is amassed to generate chips dedicated to analysis of different cancer cell types? 3) Can candidate cDNA sequences (CGAP) and larger genomic regions identified by computer analysis such as Virtual Expression Array calculations be re-sequenced to identify informative gross variations down to SNPs in cancer patient populations to identify new oncogenes or tumor suppressor genes? The specific aims of this program are: 1) To develop bioinformatics tools for the identification and ellucidation of candidate cancer related genes including software for the design, readout and analysis of gene expression and re-sequencing arrays; 2) to develop and complete a Digital Optical Chemistry (DOC) array fabrication unit capable of synthesis of at least 100,000 custom oligonucleotide array members on a single chip with the capability of rapidly constructing chips with different arrays every 2 hours; 3) to develop and construct a custom DOC readout system by modifying/replicating an in-house developed hyperspectral imaging microscope; and 4) the integration of the entire system of software and hardware for the design, fabrication, and data analysis of DNA microarray chips and their subsequent testing on clinical samples relevant for cancer research. The testing of the integrated system for use in mutation detection, SNP discovery, allelotyping, and expression analysis will progress to use archival and prospectively collected cells from biopsies and fine needle aspirates that have been purified with the new laser capture microdissection system. This will integrate our system with current major technologies to produce a tool that should be widely available in translational and clinical trials cancer research.