Nucleic Acids Isolation and DNA Analysis Facility

核心C的具体目标是： 1)LFS和WT患者淋巴母细胞系的建立和成纤维细胞的培养。 2)从外周血中提取DNA、RNA和血浆；从肿瘤和正常组织中提取DNA和RNA 研究对象的组织样本。这包括那些患有肾母细胞瘤的人，那些 潜在的p53突变携带者和这些人的家庭成员。 3)通过对P53、WT1和/或某些WT“途径”的PCR产物直接测序来检测突变 儿童肉瘤家系、Wilms肿瘤家系和散发性Wilms肿瘤的基因 4)对WT患者进行Southern分析，以确定通过基于PCR的测序无法检测到的WT1缺失。 5)奇数P53错义突变的功能特征和潜在截断的鉴定 携带无义和剪接突变体的个体细胞中的蛋白质 6)项目4中确定的WT途径基因的波分析突变筛查 7)根据需要储存和分发DNA、RNA和血浆样本 8)维护一个详细说明样本处理、核酸分离和突变的数据库 分析。该数据库将用于更新由Core C管理的程序数据库。 9)序列和突变筛选结果的存档。 这些职能对P01内所有项目的成功至关重要。项目1使用P53和WT1数据 为了更好地表征癌症风险，也为了开发更好的癌症易感性的遗传模型。这个 有关P53突变状态的信息还将识别有助于识别的非P53家族 (项目。2)负责易感性的其他基因并指导对肿瘤数据的解释 由突变小鼠产生(项目3和5)。WT1突变状态对于描述 肾母细胞瘤和WT患者的分子亚群及其生物学效应 我们以前在肾母细胞瘤中发现的突变、改变和表达变化(项目4)。 该核心提供样本处理、突变筛选和分析、功能分析、样本 存储，以及对P01所有项目至关重要的数据存储。

The specific goals for Core C are: 1) establishment of lymphoblastoid cell lines and fibroblast cultures on LFS and WT patients. 2) isolation of DNA, RNA, and plasma from peripheral blood; and DNA and RNA from tumor and normal tissue samples from study individuals. This includes those with Wilms tumors, those who are potential p53 mutation carriers, and family members of these individuals. 3) mutation detection by direct sequencing of PCR products for p53, WT1, and/or some WT "pathway" genes for childhood sarcoma kindreds, Wilms tumor kindreds, and sporadic Wilms tumors 4) Southern analysis of WT patients to identify WT1 deletions undetectable by PCR-based sequencing. 5) functional characterization of odd p53 missense mutations and identification of potentially truncated proteins in cells from individuals carrying nonsense and splicing mutants 6) mutation screening by WAVE analysis for selected WT pathway genes identified in Project 4 7) storage and distribution, as needed, of DNA, RNA, and plasma samples 8) maintenance of a database detailing sample handling, nucleic acids isolations, and mutational analysis. This database will be used to update the Program's database managed by Core C. 9) archiving of sequence and mutation screening output. These functions are critical to the success of all projects within the P01. Project 1 uses p53 and WT1 data to characterize better cancer risk and also to develop better genetic models for cancer predisposition. The information regarding p53 mutational status will also identify non-p53 families that will aid the identification (Proj. 2) of other genes responsible for predisposition and guide the interpretation of the tumor data generated from mutant mice (Projects 3 & 5). WT1 mutational status is critical for the delineation of the molecular subsets of Wilms tumors and WT patients and elucidating the biologic effect of the pattern of mutations, alterations, and expression changes we have previously identified in Wilms tumors (Project 4). This core provides sample processing, mutation screening and analyses, functional analyses, sample storage, and data storage critical for all projects of the P01.