Sequence-independent cloning methods for long DNA fragments applied to synthetic biology

Simplified methods to assemble DNA fragments by independent cloning sequence have helped in the progress of synthetic biology, allowing some biotechnological processes to become economically viable by genetic improvement of microorganisms. We compared three methods of assembling six DNA fragments: PCR fusion-based, isothermal NEBuilder and circular polymerase extension cloning (CPEC). Double and triple fusion occurs directly with the PCR products using PCR fusion-based and NEBuilder methods. For multiple fragments the results showed higher efficiency by the CPEC method which allowed assembly of six fragments previously purified by agarose gel extraction, after a sequence of 20 annealing/extension cycles without any primer.