A 13q32 BAC Microarray for chromosome function analysis

描述（由申请人提供）： 本研究的主要目的是发展基因组DNA微阵列技术，以分析非蛋白质编码功能DNA元件的生物学作用。我们将构建一个包含115个BAC的DNA微阵列，这些BAC连续跨越12.3 Mbp，覆盖人类染色体13 q32带。该13 q32 DNA微阵列将用于进一步表征7个人类细胞系，每个细胞系包含由染色体13 q的一部分的反向复制组成的额外染色体（invdup 13 q染色体）。由于在13 q32带中形成新着丝粒，这些invdupl 3q染色体中的每一个都是有丝分裂稳定的。该13 q32微阵列的功效将通过使用比较基因组杂交（CGH）来精确地定位三个invdup 13 q32染色体中的断裂点来证明。每个新着丝粒的位置将通过用染色质免疫沉淀（CHIP）获得的DNA筛选13 q32微阵列来确定，所述DNA使用针对着丝粒特异性组蛋白H3变体CENP-A的抗体获得。含有新着丝粒的BAC的鉴定将允许研究一级DNA序列在新着丝粒形成中的作用，并研究新着丝粒处着丝粒染色质结构域的基因组组织。将通过比较来自S期和G1期的不同部分的DNA的杂交来检查跨13 q32的DNA复制定时。复制时间将与13 q32的新着丝粒位置相关。因此，这个13 q32微阵列将被用来检查在人类新着丝粒形成的结构和功能的染色体特征的作用。这些类型的高密度基因组微阵列的发展将是理解我们细胞中非蛋白质编码功能DNA元件的基因组组织和作用的重要一步。

DESCRIPTION (provided by applicant): The main objective of this proposal is to develop genomic DNA microarray technology to analyze the biological role of non-protein-coding functional DNA elements. We will construct a DNA microarray that contains 115 BACs which contiguously span 12.3 Mbp covering human chromosome band 13q32. This 13q32 DNA microarray will be used to further characterize seven human cell lines that each contain a supernumerary chromosome consisting of an inverted duplication of a portion of chromosome 13q (invdup 13q chromosome). Each of these invdupl3q chromosomes is mitotically stable due to the formation of a neocentromere in band 13q32. The efficacy of this 13q32 microarray will be demonstrated by using Comparative Genomic Hybridization (CGH) to precisely map the breakpoints in three invdup13q32 chromosomes. The position of each neocentromere will be determined by screening the 13q32 microarray with DNA obtained by chromatin immunoprecipitation (CHIP) using antibodies to CENP-A, the centromere-specific histone H3 variant. Identification of BACs that contain the neocentromere will permit investigation of the role of primary DNA sequence in neocentromere formation, and investigation of the genomic organization of centromeric chromatin domains at neocentromeres. DNA replication timing across 13q32 will be examined by comparative hybridization of DNA from different fractions of S phase and from G1 phase. Replication timing will be correlated with neocentromere position across 13q32. Thus, this 13q32 microarray will be used to examine the role of structural and functional chromosome characteristics in human neocentromere formation. The development of these types of high-density genomic microarrays will be a major step towards understanding the genomic organization and role of non-protein coding functional DNA elements in our cells.