Figure 4.

Schematic representation of direct sequencing by transcriptional sequencing. (A) Thin lines are double strand templates whereas thick lines are primers. The phage promoter sequence can be appended to one or both of the PCR primers and incorporated into the PCR product. Alternatively, because most cloning vectors contain two different opposite-oriented phage promoters flanking a multiple cloning site, an inserted DNA can be amplified by using flanking primers and transcribed directly. (B) The remaining primers and 2′-dNTPs need not be removed from PCR products prior to sequencing because the transcription reaction is independent of residual primers and dNTPs. RNA fragments are transcribed under a phage promoter from unpurified PCR reactant and terminated by four kinds of dye-3′dNTPs. Sequencing products are purified and subjected to electrophoresis on a DNA sequencer. This procedure simply requires the addition of one aliquot to the PCR product.