Isolation of Full Size BAC Inserts by DNA Gap Repair in E. coli

Abstract

DNA polymers can comprise millions of base pairs and encode thousands of structural and regulatory genetic elements. Thus, the precise isolation of specific DNA segments is required for accurate gene dissection. Although polymerase chain reaction (PCR) is a standard tool for this purpose, increasing DNA template size leads to the accumulation of polymerase errors, hindering the precise isolation of large-size DNA fragments. Unlike PCR amplification, DNA gap repair (DGR) is a virtually error-free process. However, the maximal size of bacterial artificial chromosome (BAC) insert isolated so far by recombination-mediated genetic engineering (recombineering) is <90 Kilobase pairs (Kbp) in length. Here, we developed a compact bacteriophage P1 artificial chromosome (PAC) vector, and we used it to retrieve a DNA segment of 203 Kbp in length from a human BAC by DGR in Escherichia coli (E. coli). We analyzed the efficiency of DGR with repressed (recombineering-) and derepressed lambda phage red genes (recombineering+). We showed that both DGR efficiency and the percentage of PAC clones containing the expected 203 Kbp BAC insert improved with increasing size of homology arms. In recombineering+ E. coli cells and with an efficiency of electroporation of 8x109/1{micro}g pUC plasmid DNA, DGR efficiency and the percentage of correct PAC clones were about 5x10-6 and 1% for 30 bp; 6x10-6 and 30% for 40 bp; and 1.5x10-5 and 80% for 80 bp homology arms, respectively. These data show that using long homology arms and a newly developed vector, we isolated for the first time nearly a full size BAC insert with a frequency of correct clones not previously reported.