The Construction of Bacterial Artificial Chromosome (BAC) Libraries

Abstract

Cloning of exogenous DNA into bacterial artificial chromosomes (BACs) pro vides a new approach to the analysis of the genomes of higher organisms [1]. BAC libraries containing large genomic DNA inserts are important tools for positional cloning, physical mapping and genome sequencing. A number of human and plant BAC libraries have been constructed (e.g., human: [2], Arabidopsis: [3], rice: [4], sorghum: [5]). Bacterial artificial chromosome vectors utilize the rE. coli single-copy fertility plasmid and can maintain genomic DNA fragments up to 350 kb. Very little or no rearrangement of the inserts or chimerism have been observed [1, 5–7]. Other systems for the cloning of large DNA fragments have been developed. The development of yeast artificial chromosome vectors (YAC: [8]) permits cloning of fragments of greater than 500 kb. However, some disadvantages of the YAC system include a high degree of chimerism and insert rearrangement that limit its usefulness [9–11]. Systems based on the bacterial F-factor (BAC: [1]) and bacteriophage Pl (PAC: [12]) have much higher cloning efficiencies, improved fidelity, and greater ease of handling as compared to the YAC system. Table 1 shows a comparison between the two most common large DNA fragment cloning systems, YACs and BACs. Because of BAC clone stability and ease of use, the BAC cloning system has emerged as the system of choice for the construction of large insert genomic DNA libraries.