THE ELIMINATION OF PRIMER-DIMER ACCUMULATION IN PCR

We attempted to produce primer-dimers (PDs) from a variety of primers with differing types and extents of complementarity, Where PDs were pr oduced they were cloned and sequenced, We were unable to produce detec table PDs either with individual primers alone or with similar sequenc e primers even if they had 3' complementarity. These observations led to the hypothesis that a system could be developed whereby the accumul ation of PDs in a PCR may be eliminated, We demonstrate a method for t he general suppression of PD formation that uses a sequence of additio nal nucleotides (a Tail) at the 5' ends of amplimers, Tailed amplimers are present at low concentration and only participate during early cy cles of PCR. In subsequent PCR cycles, amplification is achieved using a single primer that has the same sequence as that of the Tail portio n of the early cycle primers, here we refer to this as a Tag. When pro ducts are small, as with PDs, there is a high local concentration of c omplementary sequences derived from the Tail, This favours the anneali ng of the complementary ends of a single strand produced by tailed pri mer interactions and gives rise to 'pan-handle' structures, The format ion of these outcompetes the annealing of further Tag primers thereby preventing the accumulation of non-specific PD products, This aids the design of large multiplex reactions and provides a means of detecting specific amplicons directly in the reaction vessel by using an interc alating dye.