Lambda‐PCR for precise DNA assembly and modification

Lambda‐polymerase chain reaction (λ‐PCR) is a novel and open‐source method for DNA assembly and cloning projects. λ‐PCR uses overlap extension to ultimately assemble linear and circular DNA fragments, but it allows the single‐stranded DNA (ssDNA) primers of the PCR extension to first exist as double‐stranded DNA (dsDNA). Having dsDNA at this step is advantageous for the stability of large insertion products, to avoid inhibitory secondary structures during direct synthesis, and to reduce costs. Three variations of λ‐PCR were created to convert an initial dsDNA product into an ssDNA “megaprimer” to be used in overlap extension: (i) complete digestion by λ‐exonuclease, (ii) asymmetric PCR, and (iii) partial digestion by λ‐exonuclease. Four case studies are presented that demonstrate the use of λ‐PCR in simple gene cloning, simultaneous multipart assemblies, gene cloning not achievable with commercial kits, and the use of thermodynamic simulations to guide λ‐PCR assembly strategies. High DNA assembly and cloning efficiencies have been achieved with λ‐PCR for a fraction of the cost and time associated with conventional methods and some commercial kits.