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.