Schematic for cDNA synthesis by template-switching.

(Step 1) Primer binding and initiation of polymerization. (Step 2) MMLV reverse transcriptase adds deoxycytosines to the cDNA 3' end. (Step 3) Template-switch oligo binds the CCC overhang. (Step 4) Reverse transcriptase switches templates and continues polymerization using the template-switch oligo as the template. (Steps 5–7) The single-stranded cDNA product of reverse transcription becomes the template for second-strand synthesis primed by the universal PCR forward primer. Amplification follows using the universal PCR forward primer and nested chain-specific PCR reverse primers. Note that the lengths of the different antibody regions and primers are not drawn to scale.