PHYSICAL MAPPING OF THE SHORT ARM OF CHROMOSOME 11

点击翻译按钮获取中文摘要

The goal of this project is to obtain a high resolution physical map of the short arm of chromosome 11. To date, chromosome 11 and X are the best characterized, with the maximum number of probes and the extent covered by overlapping clones. As such, the strategies developed here could serve as a paradigm for the mapping other regions of the genome. Our strategy consists of preparing chromosome 11 libraries in yeast artificial chromosomes (YAC) using DNA from flow sorted chromosome 11 purified from somatic cell hybrids containing this chromosome as its only human material. YAC clones will be arrayed in 96-well plates and archived, the name of each clone being assigned according to its grid coordinates. Identification of YAC clones harboring genes and markers mapping to the short arm, following PCR analysis/hybridization/screening of clones by the multiplex procedure will reveal the extent of the chromosome 11p representation in the library. The characterization of YAC clones for presence and size human DNA insert, mapping of rare restriction sites, the identification of sequences at the extreme ends by inverse PCR of circularized YAC fragments, and in situ hybridization of YAC clones to metaphase chromosomes directly or following subcloning into cosmids, will assist in determining orientation of the clones and in construction of contigs. We will need a set of 100 YAC clones with an average of 500 kb inserts, and 1250 cosmid clones with 40 kb inserts, aligned end to end, to span the roughly 50 mb short arm of chromosome 11. Further detailed analysis of genes or loci, mapping of regions harboring potential disease genes, their cloning and characterization will be carried out in cosmid subclones. Sequencing of double stranded cosmid DNA template using the T7 or T3 promoter sites, present in the cosmid vector will generate sequence tagged site (STS) for each of the cosmid clones, a reference point for the progress of the project as well as providing an approach for map closure. By using a combination of YAC cloning which we will embark upon, and the cosmid clones to be generated in Project 1, we aim to provide a physical map of the short arm with spacing of landmark probes at about 300 kb apart. In addition, this project will be investigating, in detail, a region of chromosome 11p15.5 to pter that has a great paucity of markers and localized genes, followed later by studies into the region from the centromere to 11p15.5. We hope to unravel genes important in health and disease, produce a detailed physical map of the short arm of chromosome 11, and produce templates for DNA sequencing.