JCLIUV2N2A4-Pan.pdf

The Cloning and Propagation of an Inverted Repeat DNAin M13 Bacteriophage in Escherichia coli

Xuefeng Pan*,^1,2,3 Nan Jiang², Liang Ding¹ and Fei Duan¹

1,School of Basic Medicine, Hebei University, Baoding 071002, China

2, School of Life Science, Beijing Institute of Technology, Beijing 100081, China

3, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080

China

Abstract

DNA inverted repeats are abundant in all genomes with potential of adopting DNA hairpin and cruciform structures that sometimes raise genomic instabilities. This work showed that an inverted repeat DNA sequence in bovine prochymosin gene could only be cloned in M13 bacteriophage by one orientation, and propagation of this orientation in E.coli cells raised an immediate deletion of a 102-bp DNA fragment, containing partial repeat DNA and partial M13 DNA. The deletion was found to be mediated by two tandemly arranged “CTGC”sequences, one in the inverted repeat sequence and another in the M13 phage, respectively, indicating that the deletion occurred via a nascent strand slipped mis-pairingreplication mechanism. We were failed in cloning the opposite orientation of the inverted repeats by screening a large number of white bacteriophage plagues, suggesting that orientation may be inviable. By contrast, the same inverted repeats can well be cloned and propagated in a pUC19 plasmid in E.coli with two orientations. These results suggested that the maintenance of the inverted repeat in vivo was tightly related to the repeat foldability which is subjected to where it is located and how it is replicated.

Key words: DNA Inverted repeat; Interspersed dinucleotide repeats; Replication

Slippage; DNA folding