A bacterial artificial chromosome library for sequencing the complete human genome

• Published in: Genome research Vol. 11; no. 3; pp. 483 - 496
• Main Authors: Osoegawa, K, Mammoser, A G, Wu, C, Frengen, E, Zeng, C, Catanese, J J, de Jong, P J
• Format: Journal Article
• Language: English
• Published: United States Cold Spring Harbor Laboratory Press 01.03.2001
• Subjects: Chromosomes, Artificial, Bacterial - genetics; Cloning, Molecular - methods; Contig Mapping; Deoxyribonuclease EcoRI - metabolism; Deoxyribonucleases, Type II Site-Specific - metabolism; DNA, Satellite - genetics; Gene Rearrangement; Genetic Markers - genetics; Genomic Library; Human Genome Project; Humans; Molecular Sequence Data; Recombinant Fusion Proteins - genetics; Recombination, Genetic - genetics; Resources; Restriction Mapping; Sequence Analysis, DNA - methods; Sequence Tagged Sites
• ISSN: 1088-9051; 1549-5469
• DOI: 10.1101/gr.169601

A 30-fold redundant human bacterial artificial chromosome (BAC) library with a large average insert size (178 kb) has been constructed to provide the intermediate substrate for the international genome sequencing effort. The DNA was obtained from a single anonymous volunteer, whose identity was protected through a double-blind donor selection protocol. DNA fragments were generated by partial digestion with EcoRI (library segments 1--4: 24-fold) and MboI (segment 5: sixfold) and cloned into the pBACe3.6 and pTARBAC1 vectors, respectively. The quality of the library was assessed by extensive analysis of 169 clones for rearrangements and artifacts. Eighteen BACs (11%) revealed minor insert rearrangements, and none was chimeric. This BAC library, designated as "RPCI-11," has been used widely as the central resource for insert-end sequencing, clone fingerprinting, high-throughput sequence analysis and as a source of mapped clones for diagnostic and functional studies.