Optimizing Megaprimer PCR of Whole Plasmids via Construction of Chimeric Membrane Proteins

Polymerase Chain Reaction (PCR) has long been the choice biochemical technique for exponentially amplifying limited copies of DNA, as well as for making mutations and modifications to existing DNA sequences. Recently, a new method of ligation-independent PCR was introduced that proved to increase the efficiency and overall clone-success rate for construction of sophisticated chimeric proteins in vitro.  Megaprimer PCR of whole plasmids (MEGAWHOP) allows large sections of genes, with the support of a megaprimer, to be mutated in one step without the use of restriction enzymes or ligation reactions. This method has also been shown to be a more efficient approach to insert random mutagenic stretches into defined segments of genes. MEGAWHOP further surpasses error-prone Quick Change mutagenesis by allowing multiple mutations of up to hundreds of base pairs to be made simultaneously. In the realm of these results, 12 protein chimeras were constructed via individually optimized MEGAWHOP reactions for the ion channel proteins TRPM8 and TRPM2. The 6 helical transmembrane regions in either protein were swapped to produce chimeric genes that will ultimately be useful for electrophysiology studies of TRP channel function. The number and viability of the clones generated were measured quantitatively and qualitatively after several reaction parameters were varied across the 12 different samples. Given the number of chimeras ultimately obtained, careful analysis indicated that certain optimizations significantly increased the odds of success for generating multiple mutations in DNA in a few steps. Efficacious optimizations included lengthening primer sequences, modifying conventional thermal cycler reaction times, and adding detergent additives to the PCR reaction mixture to produce clones with minimized unintended mutations in significantly greater quantities than the standard MEGAWHOP protocol (yields increased, on average, by 56%). These results ultimately validate the advantages of MEGAWHOP over traditional PCR, as well as provide a basis for more efficient MEGAWHOP practice in future cloning and expression experiments.