Fig 3. Producing two single-stranded ends by incomplete PCR.

If primer extension in late PCR cycles is incomplete in a subset of PCR products, double-stranded DNA molecules with a single-stranded end may form. To produce double-stranded DNA molecules with two single-stranded ends, the PCR mixtures have to be denatured after the final polymerase extension step to allow new strand combinations upon renaturation. The concept shown here for the plasmid also applies to the insert. By the same mechanism, double-stranded DNA molecules containing two single-stranded ends could potentially form during PCR cycling if critical reagents are used up prior to the last PCR cycle(s), so that no DNA synthesis takes place in the last cycle(s).