DNA ‘Breathing’ Recombination Cloning: A Mismatch-Tolerant, Temperature-Dependent Homologous Recombination Cloning Method

DNA cloning traditionally relies on two approaches: restriction endonuclease digestion-ligation, and homologous recombination involving exonucleases, polymerases, and other enzymes. Here, we present a novel cloning method that requires only restriction endonucleases, eliminating the need for exonucleases or polymerases. The linearized cloning vector and the foreign DNA fragment (FDF) containing overlapping sequences were mixed and incubated at the melting temperature of the overlapping DNA sequences for 5 min, then cooled slowly to 0 °C. The mixture was transformed into <i>E. coli</i> and positive transformants were obtained. This cloning method was named DNA 'breathing' recombination (DBR) cloning. The overlapping sequence between the linearized vector and the FDF is preferably from 12 to 16 base pairs. Even when the ends of the linearized vector contain mismatches of up to 20 base pairs with the ends of the FDF, the DBR cloning method can still proceed efficiently, enabling truly seamless assembly. Meanwhile, the DBR method supports one-step assembly of multiple fragments. Therefore, the DBR cloning method simplifies experimental operations and reduces experimental costs while maintaining high cloning efficiency.