PHYSICAL & REFINED GENETIC MAP OF HUMAN CHROMOSOME 10

描述：(改编自调查人员摘要)长期 这项研究的目标是对人类10号染色体进行测序。 这项提议的具体目标是构建一张完整的物理地图 10.含有大DNA片段的酵母人工染色体 将被鉴定，然后按照高分辨率的基因排序 地图，它将被构建为本提案的一个具体目标。这个 构成这一物理地图的有序克隆将成为 对染色体进行测序。具体地说，10号染色体YAC文库将 是被建造的。这个库将由1,500个克隆组成，这些克隆 平均插入大小为350kb，将构成相当于3个 10号染色体的拷贝。找到一种特定的 10号染色体的序列在这样的集合中占95%。在适当的时候， 来自YAC的亚克隆将被制作并筛选多态，这将 被定位在大家族上，以提炼先前存在的基因 地图。染色体行走将通过基于STS/PCR的方法来扩展基因座 策略。单个YAC的末端片段将通过以下方式扩增 反向聚合酶链式反应，并将开发STSS。为了识别重叠的YAC，聚合酶链式反应 将在YAC池上执行STSS，然后在以下池的子池上执行 测试呈阳性。YAC文库中STSS的多重系统筛查 将允许不同克隆中的连续序列快速 订好了。为了帮助订购YAC重叠群，单个YAC将被分配到 利用一组特征良好的杂交细胞系研究染色体区域。 包含与映射的RFLP探针同源的序列的YAC将 作为轨迹扩展的成核点，因为相对的 这样的探头的位置是明确的。重叠群的方向 将根据精细的遗传图谱进行推断。差距 YAC之间的重叠群将首先通过走在COSMID库中来填充。 剩余差距的性质和程度将由长期来决定 限制映射。这些空白将由额外的YAC来填补 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.