DNA assembly sequences into plasmids is one of the most important basic technologies for bioscience research and metabolic engineering. There are many of molecular cloning techniques have been developed and these techniques that need specialized expensive reagents or laborious experimental procedure. For that reason, a significant amount of effort has been dedicated to developing better DNA assembly methods with higher efficiency and fidelity as well as simpler and faster protocols. Here, we compared between conventional, in vivo and in vitro DNA assembly methods and their recent applications, we also highlight the optimum protocol for in vivo cloning of DNA assembly methods. The present study concluded that vector construction can be carried out simply by simply placing a DNA fragment having a homologous sequence and directly transformed into E. coli and this method gives great help in improving efficiency of molecular biological research