Schematic illustrations of the preparation of double-stranded DNA template for in vitro RNA transcription. (A) Preparation of a DNA template from plasmid DNA. A target DNA fragment harboring the T7 promoter sequence (T7 pro) at the 5′ end of the gene is prepared from plasmid DNA by linearization using a restriction enzyme or by PCR amplification. (B) Three distinct strategies to prepare a DNA template from two oligonucleotides. Strategy 1: A double-stranded DNA template is prepared by annealing nontemplate strand (NTS) and template strand (TS) DNA oligonucleotides encoding the T7 pro sequence and the entire target gene. Strategy 2: A hemi-duplexed DNA template is prepared by annealing a NTS oligonucleotide encoding the T7 pro sequence to a TS oligonucleotide encoding the T7 pro and the entire gene. Strategy 3: Two oligonucleotides are designed to possess an overlapping region that facilitates their annealing and are extended by DNA polymerase.
