PHYSICAL & REFINED GENETIC MAP OF HUMAN CHROMOSOME 10

描述：（改编自研究者摘要）长期 本研究的目的是对人类10号染色体进行测序。 的 这项建议的具体目标是建立一个完整的物理地图， 10号染色体。 含有大DNA片段的酵母人工染色体 将被识别，然后按照高分辨率遗传 地图，这将是本提案的一个具体目标。 的 构成这张物理图谱的有序克隆将是DNA的来源， 测序染色体。 具体地，染色体10 YAC文库将 被建造。 这个文库将由1,500个克隆组成， 平均插入片段大小为350 kb，将构成相当于三个 10号染色体的拷贝。 找到一个特定的 10号染色体序列在这样的集合中是95%。 在适当的时候， 将从YAC制备亚克隆并筛选多态性， 在大家庭中进行映射，以改进先前存在的遗传 地图 染色体步移将用于通过基于STS/PCR的扩增基因座。 战略 单个YAC的末端片段将通过PCR扩增。 将开发反向PCR和STS。 为了鉴定重叠的YAC，PCR 的STS将在YAC池上执行，然后在 检测呈阳性 YAC文库中STS的多重系统筛选 将允许不同克隆中的连续序列快速地 命令。 为了帮助排序YAC重叠群，将单个YAC分配给 使用一组充分表征的杂交细胞系对染色体区域进行分析。 含有与定位的RFLP探针同源的序列的YAC将 可以用作轨迹扩展的成核点，因为相对的 这些探针的位置被很好地限定。 重叠群的方向 将根据精细的遗传图谱推导出染色体上的基因。 差距 将首先通过在粘粒文库中步移来填充YAC重叠群之间的序列。 剩余差距的性质和程度将由长期 限制性酶切作图 这些空缺将由更多的青年咨询委员会填补 克隆。

DESCRIPTION: (Adapted from Investigators' Abstract) The long term objective of this research is to sequence human chromosome 10. The specific goal of this proposal is to construct a complete physical map of chromosome 10. Yeast artificial chromosomes containing large DNA fragments will be identified and then ordered following a high resolution genetic map, which will be constructed as a specific goal of this proposal. The ordered clones constituting this physical map will be the source of DNA for sequencing the chromosome. Specifically, a chromosome 10 YAC library will be constructed. This library, which will consist of 1,500 clones whose average insert size is 350 kb, will constitute the equivalent of three copies of chromosome 10. The probability of finding a particular chromosome 10 sequence in such a collection is 95%. When appropriate, subclones from YACs will be made and screened for polymorphisms which will be mapped on extended families so as to refine the pre-existing genetic map. Chromosome walking will be used to expand loci by an STS/PCR-based strategy. The end fragments of individual YACs will be amplified by inverse PCR, and STSs will be developed. To identify overlapping YACs, PCR of STSs will be performed on YAC pools and then subpools of those which test positive. Multiple, systematic screening for STSs in the YAC library will allow the contiguous sequences in different clones to be rapidly ordered. To help order YAC contigs, individual YACs will be assigned to chromosomal regions using a well-characterized panel of hybrid cell lines. YACs containing sequences which are homologous to mapped RFLP probes will be used as nucleation points for locus expansion since the relative positions of such probes are well defined. The orientation of the contigs on the chromosome will be deduced following the fine genetic map. Gaps between YAC contigs will first be filled by walking in cosmid libraries. The nature and extent of remaining gaps will be determined by long range restriction mapping. These gaps will then be filled by additional YAC cloning.