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Lambda-PCR for precise DNA assembly and modification
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DOI：10.1002/bit.28240.png)
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Lambda-polymerase chain reaction (lambda-PCR) is a novel and open-source method for DNA assembly and cloning projects. lambda-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 lambda-PCR were created to convert an initial dsDNA product into an ssDNA megaprimer to be used in overlap extension: (i) complete digestion by lambda-exonuclease, (ii) asymmetric PCR, and (iii) partial digestion by lambda-exonuclease. Four case studies are presented that demonstrate the use of lambda-PCR in simple gene cloning, simultaneous multipart assemblies, gene cloning not achievable with commercial kits, and the use of thermodynamic simulations to guide lambda-PCR assembly strategies. High DNA assembly arid cloning efficiencies have been achieved with lambda-PCR for a fraction of the cost and time associated with conventional methods and some commercial kits.
Keyword:
DNA assembly
DNA modifications
PCR
recombinant plasmids
lambda-exonuclease
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3.6
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2.2W
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