FULL LENGTH CDNA SEQUENCING FOR PEDIATRIC LEUKEMIAS

描述：(申请者描述)该项目旨在生成 来自新鲜的儿童白血病细胞的文库已经直接 确诊时从血液中分离出来的。代表 儿童急性淋巴细胞白血病(ALL)的主要亚型和至少一种 将选择急性髓系白血病(AML)亚型进行分析。 图书馆建设方法将利用现有的改进 已演示的生成库的技术具有较高的 箔长克隆的比例。这些库中的克隆将包含 原始mRNA的全长拷贝，文库将是 原始信使群体的代表。 这些文库将被标准化，以减少丰富的 来自非标准化文库和标准化文库的不同克隆和等位基因 将排列成阵列，以便于复制和识别 利息。将对10,000个随机选择的克隆进行EST测序 每年执行以选择看起来全长的DNA，并用于 在数据库中没有现有的箔长度序列可用。 将分别确定3501.5-2.0kb cDNA全长序列 年，使用一种串联cdna测序方法，该方法是在 这个实验室。该协议使我们能够同时执行 构建单个DNA后对多达70个cDNA进行全长测序 猎枪图书馆。一种基于聚合酶链式反应的优化方法 实验室将用于识别未满的克隆的5‘末端 长度。所有数据将立即发布，所有克隆都将自由 可用。 这些恶性组织还没有被广泛测序，可以 因此，预计将提供大量新信息，与 是从白血病细胞系中提取的。因此，这些产品的可用性 图书馆将提供一种快速隔离全长副本的机制 之前识别的部分克隆，并将为 识别新的癌症相关基因。组织的整合 收藏、文库建设、EST分析、全长克隆插入 测序和5‘端救援将定义一条完整且可扩展的途径 对与癌症有关的基因进行系统分析。

DESCRIPTION: (Applicant's Description) This project aims to generate cDNA libraries from fresh pediatric leukemia cells that have been directly isolated from blood at the time of diagnosis. Cases that represent the major subtypes of childhood Acute Lymphoid leukemia (ALL) and at least one subtype of Acute Myelogenous Leukemia (AML) will be chosen for analysis. The library construction methodology will utilize improvements on existing techniques which have been demonstrated to generate libraries with a high proportion of foil-length clones. Clones from these libraries will contain full-length copies of the original mRNA, and the libraries will be representative of the original messenger population. The libraries will be normalized to reduce the variation in abundance of different clones and aliquots from non-normalized and normalized libraries will be arrayed to facilitate replication and identification of clones of interest. EST sequencing on 10,000 randomly selected clones will be performed each year to select cDNAs that appear to be full- length, and for which there is no existing foil length sequence available in the databases. The full length sequence of >350 1.5-2.0 kb cDNAs will be determined each year, using a concatenation cDNA sequencing approach that was developed in this laboratory. This protocol enables us to simultaneously perform full-length sequencing on as many as 70 cDNAs after construction of a single shotgun library. A PCR-based method that has been optimized m our laboratory will be used to identify the 5'-ends of clones that are not full length. All data will be released immediately and all clones will be freely available. These malignant tissues have not been extensively sequenced, and can therefore be expected to provide substantial new information compared to that derived from leukemia cell lines. Hence, the availability of these libraries will provide a mechanism for rapidly isolating full-length copies of previously identified partial clones, and will provide a source for identifying new cancer-related genes. The integration of the tissue collection, library construction, EST analysis, full length clone insert sequencing and 5'- end rescue will define a complete and scalable pathway for the systematic analysis of genes involved in cancer.