FULL LENGTH CDNA SEQUENCING FOR PEDIATRIC LEUKEMIAS

描述：（申请人的描述）本项目旨在产生cDNA 从新鲜的儿科白血病细胞中提取的文库， 在诊断时从血液中分离出来。 案件代表了 儿童急性白血病（ALL）的主要亚型和至少一种 将选择急性骨髓性白血病（AML）亚型进行分析。 图书馆建设方法将利用现有的 已被证明可以产生具有高 叶长克隆的比例。 这些库中的克隆将包含 原始mRNA的全长拷贝，并且文库将 代表了最初的信使人口。 将文库标准化以减少以下丰度的变化： 来自非标准化和标准化文库的不同克隆和等分试样 将被排列，以便于复制和鉴定克隆的 兴趣 将对10，000个随机选择的克隆进行EST测序， 每年进行一次，以选择似乎是全长的cDNA， 在数据库中没有现有的箔长度序列。 将测定>350个1.5-2.0 kb cDNA的全长序列， 年，使用一种串联cDNA测序方法， 这个实验室。 该协议使我们能够同时执行 在构建一个单一的cDNA序列后， 猎枪图书馆 一种基于PCR的方法，已被优化，我们 实验室将用于鉴定克隆的5 '-末端， 长度 所有数据将立即公布，所有克隆将自由 available. 这些恶性组织还没有被广泛测序， 因此，预计将提供大量的新信息相比， 来源于白血病细胞系。 因此，这些可用性 库将提供一种快速分离全长拷贝的机制 以前确定的部分克隆，并将提供一个来源， 发现新的癌症相关基因。 组织的整合 文库构建，EST分析，全长克隆插入 测序和5 '-末端拯救将定义一个完整的和可扩展的途径 用于系统分析癌症相关基因。

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.