The StrataClone PCR Cloning Kit allows high-efficiency, 5-minute cloning of PCR products, using the efficient DNA rejoining activity of DNA topoisomerase I and the DNA recombination activity of Cre recombinase.
Overview of StrataClone PCR Cloning Technology
Using the method summarized in Figure 1, StrataClone PCR cloning technology exploits the combined activities of topoisomerase I from Vaccinia virus and Cre recombinase from bacteriophage P1. In vivo, DNA topoisomerase I assists in DNA replication by relaxing and rejoining DNA strands. Topoisomerase I cleaves the phosphodiester backbone of a DNA strand after the sequence 5´-CCCTT, forming a covalent DNA–enzyme intermediate which conserves bond energy to be used for religating the cleaved DNA back to the original strand. Once the covalent DNA–enzyme intermediate is formed, the religation reaction can also occur with a heterologous DNA acceptor. The Cre recombinase enzyme catalyzes recombination between two loxP recognition sequences.
The StrataClone PCR cloning vector mix contains two DNA arms, each charged with topoisomerase I on one end and containing a loxP recognition sequence on the other end. The topoisomerase-charged ends have a modified uridine (U*) overhang. Taq-amplified PCR products, which contain 3´-adenosine overhangs, are efficiently ligated to these vector arms in a 5-minute ligation reaction, through A-U* base-pairing followed by topoisomerase I-mediated strand ligation.
The resulting linear molecule (vector armori–PCR product–vector armamp/kan) is then transformed, with no clean-up steps required, into a competent cell line engineered to transiently express Cre recombinase. Cre-mediated recombination between the vector loxP sites creates a circular DNA molecule (pSC-A-amp/kan, see Figure 2) that is proficient for replication in cells growing on media containing ampicillin or kanamycin. The resulting pSC-A-amp/kan vector product includes a lacZ´ a-complementation cassette for blue-white screening.
Figure 1: Overview of the StrataClone PCR cloning method