FIGURE 1.

Schematic diagram showing theoretical improvement of asymmetric PCR yield by the use of a blocking primer. (A) Regular asymmetric PCR. The forward strands (FS, solid black lines) compete with the forward primer (FP, short gray lines) for the binding to the reverse strands (RS, dashed lines). In early cycles of amplification the competitiveness of FP (gray sphere) and FS (black sphere) are comparable due to the high concentration of FP and the length of FS. This will lead to equiprobable binding to the RS. With further cycles the quantity of FP decreases and the quantity of FS increases. This will lead to an increasing competiveness of FS however the competitiveness of FP will drop resulting in no more binding of FP to RS. In other words generation of FS will be blocked. Under such conditions the plateau of amplification of single stranded FS will be reached at very early cycles. (B) Asymmetric PCR with dideoxy reverse primer (DDRP). Addition of DDRP (short dashed gray lines with gray circle at the end) to the PCR reaction will limit the competitiveness of FS enhancing the binding of FP to RS which leads to the generation of higher quantity of single stranded FS. Under such condition the plateau of amplification of single stranded FS will be reached at later cycles.