Genetic Analysis of Amplified Genomic DNA Archives

描述（由申请人提供）： 现代DNA分析方法和微阵列技术是潜在的工具，用于肿瘤的综合遗传分析以及处于癌症风险的患者的癌前病变。然而，这种分析的一个重大障碍是需要相对较大的DNA样品。最近开发的等温全基因组扩增方法有望消除这些障碍，全面的遗传分析。该研究项目旨在展示简单而强大的扩增程序的实用性，用于从小组织样本中生成基因组DNA的存档拷贝。头颈癌将被用作模型，以优化和验证肿瘤和肿瘤前病变的DNA存档程序。将前瞻性采集样本，并采集癌组织、癌组织边缘以及其他被确定为肿瘤前病变候选者的病变。将使用激光捕获显微切割分离样本子集。将扩增所有采集的组织样本和显微切割样本，以生成头颈癌和癌前病变样本的DNA档案。各种方法，包括微卫星分析，检测人乳头瘤病毒亚型，和比较基因组杂交（CGH）的BAC微阵列，将被用来调查和验证的效用档案扩增DNA。BAC微阵列上的CGH分析能够检测存档DNA样本中数千个基因位点的遗传改变，这些基因位点代表癌症、癌前病变或良性发育不良的不同阶段。生物信息学工具将用于构建基于距离树或聚类算法的替代分类方案，利用微卫星，阵列CGH和HPV感染状态观察的完整数据集。本研究的目的是作为一个示范和验证的效用的DNA档案，产生的等温全基因组扩增，在癌症遗传学的综合研究。本研究建立的新能力应可扩展到任何人类癌症模型，并应通过消除与有限的生物材料可用性相关的许多限制，大大扩展在整个基因组水平上的遗传分析的利用。

DESCRIPTION (provided by applicant): Modern DNA analytical methods and microarray technologies are potentially enabling tools for the comprehensive genetic analysis of tumors as well as premalignant lesions in patients at risk for cancer. A significant obstacle to such analysis, however, is the need for relatively large DNA samples. A recently developed isothermal whole genome amplification method promises to eliminate these barriers to comprehensive genetic analysis. This research project aims to demonstrate the utility of simple and robust amplification procedures for generating archival copies of genomic DNA from small tissue samples. Head and neck cancer will be used as a model to optimize and validate procedures for DNA archiving of neoplastic and pre-neoplastic lesions. Samples will be collected prospectively, and cancer tissue, cancer tissue margins, as well as other lesions identified as candidates for pre-neoplasia will be collected. A subset of the samples will be isolated using laser capture microdissection. All collected tissue samples and microdissected samples will be amplified to generate a DNA archive of head and neck cancer and premalignant lesion specimens. A variety of methods, including microsatellite analysis, detection of human papillomavirus subtypes, and comparative genomic hybridization (CGH) on BAC microarrays, will be used to investigate and validate the utility of the archive of amplified DNA. CGH analysis on BAC microarrays enables the detection of genetic alterations at thousands of gene loci in the archived DNA samples, which are representative of different stages of cancer, preneoplasia, or benign dysplasia. Bioinformatics tools will be used to construct alternative classification schemes based on distance-based trees or clustering algorithms, utilizing the complete data set of microsatellite, array-CGH, and HPV infection status observations. This study is intended to serve as a demonstration and validation of the utility of DNA archives, generated by isothermal whole genome amplification, for comprehensive studies in cancer genetics. The novel capabilities established by this study should be extensible to any human cancer model and should greatly expand the utilization of genetic analysis at the level of the entire genome by eliminating many of the constraints related to limited availability of biological material.