Preparation of long templates for RNA in vitro transcription by recursive PCR

Preparing conventional DNA templates for in vitro RNA transcription involves PCR amplification of the DNA gene coding for the RNA of interest from plasmid or genomic DNA, subsequent amplification with primers containing a 5' T7 promoter region, and confirmation of the amplified DNA sequence. Complications arise in applications where long, nonnative sequences are desired in the final RNA transcript. Here we describe a ligase-independent method for the preparation of long synthetic DNA templates for in vitro RNA transcription. In Recursive PCR, partially complementary DNA oligonucleotides coding for the RNA sequence of interest are annealed, extended into the full-length double-stranded DNA, and amplified in a single PCR. Long insertions, mutations, or deletions are accommodated prior to in vitro transcription by simple substitution of oligonucleotides.