Lambda-PCR for precise DNA assembly and modification

Abstract

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.

Keywords: DNA assembly; DNA modifications; PCR; recombinant plasmids; λ-exonuclease.

Figure 1

Schematic of the lambda‐polymerase chain reaction (λ‐PCR) v1 protocol, which uses complete digestion with λ‐exonuclease to generate the ssDNA megaprimer. (a) PCR amplification of target gene with the phosphorylated reverse primer. (b) λ‐exonuclease complete digestion of the reverse strand of the PCR fragment. (c) ssDNA megaprimer. (d) Annealing of megaprimer and plasmid reverse primer. (e) First PCR cycle and amplification of the plasmid. Dashed strands are newly amplified. (f) Second PCR cycle and plasmid amplification. (g) Third cycle of PCR and amplification of the target plasmid.

Figure 2

Schematic of the lambda‐polymerase chain reaction (λ‐PCR) v2 protocol, which uses asymmetric PCR to generate the ssDNA megaprimer. (a) PCR amplification of target gene with excess forward primer. (b) ssDNA megaprimer. (c) Annealing of megaprimer and plasmid reverse primer. Dashed strands are newly amplified. (d) First PCR cycle and amplification of the plasmid. (e) Second PCR cycle and plasmid amplification. (f) Third cycle of PCR and amplification of the target plasmid.

Figure 3

Schematic of the lambda‐polymerase chain reaction (λ‐PCR) v3 protocol, which uses partial digestion with λ‐exonuclease to generate the dsDNA megaprimer with ssDNA overhangs. (a) PCR amplification of target gene with phosphorylated forward and reverse primer. (b) λ‐exonuclease partial digestion of the PCR fragment. (c) dsDNA megaprimers. (d) Annealing of megaprimers and plasmid. PCR amplification to incorporate the megaprimers into a dsDNA fragment. (e)  Ligation to circularize the target plasmid.