Fig. 1

A schematic representation of the LREI cloning procedure. The technique involves designing primers that incorporate the gene specific untranslated region (UTR) (yellow) and nucleotides homologous to the plasmid pHW2000 (red) multiple cloning site (MCS) to the polymerase coding region resulting in formation of a megaprimer. The viral RNA extracted from the influenza virus (1) can be reverse transcribed by using universal 12 primers (AGCAAAAGCAGG) (2), and the megaprimer can be amplified either from cDNA or any donor plasmid using the primers mentioned in Table 1 (3). Denaturation of the megaprimer generates two primers having complementary ends to the pHW2000 MCS, which when used with a bait plasmid facilitate annealing (4,5). Subsequent thermocycling steps result in extension of the annealed primers (5) thereby synthesizing the non-methylated DNA insert along with the pHW2000 vector as a mixture. DpnI treatment results in digestion of parental methylated DNA, (7) leaving the newly synthesized non-methylated DNA, which can be transformed into E. coli resulting in formation of the desired recombinant plasmid (8)