MOLECULAR BIOLOGY

核心A是该计划的分子生物学组成部分。它将具有四项重要职能： 1)测试所有活检和眉毛样本是否存在β HPV DMA。2)检测血清 β HPV抗体的存在。3)开发和维护计算机化数据库， 跟踪组织样本和小鼠，生成工作列表，并将组织学和实验室测试结果与 个体小鼠或样本。4)存档质粒、细胞系和逆转录病毒。此外，该核心还具有 开发组件，以建立一个多重的，高通量的测定，以检测HPV DNA。 为了检测β HPV DNA，我们将使用一组来自 E1开放阅读框中的保守区。扩增的DMA将被克隆和测序（10 克隆/样品）。将使用Blast检索将HPV识别为已知或新型HPV。为 为了检测HPV抗体，我们将采用我们实验室最近建立的方法 它能够同时检测多种α、β、γ和μ HPV类型的抗体， 并以高度敏感和特异的方式进行。该方法使用Luminex技术，其中GST- L1蛋白偶联至有色聚苯乙烯珠。将珠子与血清沿着混合， 标准检测试剂，Luminex读取与每个微珠结合的抗体量。 这个核心的最大优势是实验室人员，外部专家已经同意， 与我们协商测试协议的开发和实施，以及优秀的设施 在弗雷德哈钦森癌症中心有售。所有核心A人员都有良好的业绩记录， 在本核心所需的工作中具有丰富的经验。

Core A is the molecular biology component of the Program. It will have four important functions: 1) Test all biopsies and eyebrow hair samples for the presence of beta HPV DMA. 2) Test sera for the presence of antibodies to beta HPVs. 3) Develop and maintain computerized databases that will be used to track tissue samples and mice, to generate work lists and to link histology and laboratory test results with individual mice or specimen. 4) Archive plasmids, cell lines and retroviruses. In addition this Core has a developmental component to establish a multiplex, high-throughput assay to detect HPV DMAs. For the detection or beta HPV DMAwe will be amplifying beta HPV DMA using a set of primers from a conserved region in the E1 open reading frame. The amplified DMAwill be cloned and sequenced (10 clones per sample). Blast searches will be use to identify the HPVs as known or novel HPVs. For the detection of antibodies to HPVs, we will adopt a method that has recently been established in our laboratory that has the ability to test for antibodies to numerous alpha, beta, gamma and mu HPV types simultaneously, and to do so in a highly sensitive and specific fashion. This method uses Luminex technology in which GST- LI proteins are coupled to colored polystyrene beads. The beads are mixed together, along with serum and a standard detection reagent, and the Luminex reads the amount of antibody bound to each bead. The greatest strengths of this Core are the laboratory personnel, the outside experts that have agreed to consult with us on the development and implementation of testing protocols, and the excellent facilities available at the Fred Hutchinson Cancer Center. All of Core A personnel have strong track records and extensive experience in the work required for this Core.