The elimination of primer-dimer accumulation in PCR

Abstract

We attempted to produce primer-dimers (PDs) from a variety of primers with differing types and extents of complementarity. Where PDs were produced they were cloned and sequenced. We were unable to produce detectable PDs either with individual primers alone or with similar sequence primers even if they had 3'complementarity. These observations led to the hypothesis that a system could be developed whereby the accumulation of PDs in a PCR may be eliminated. We demonstrate a method for the general suppression of PD formation that uses a sequence of additional nucleotides (a Tail) at the 5' ends of amplimers. Tailed amplimers are present at low concentration and only participate during early cycles of PCR. In subsequent PCR cycles, amplification is achieved using a single primer that has the same sequence as that of the Tail portion of the early cycle primers, here we refer to this as a Tag. When products are small, as with PDs, there is a high local concentration of complementary sequences derived from the Tail. This favours the annealing of the complementary ends of a single strand produced by tailed primer interactions and gives rise to 'pan-handle' structures. The formation 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 intercalating dye.

Development of a quantitative real-time RT-PCR for kinetic analysis of immediate-early transcripts of Rat cytomegalovirus

“…A melting curve analysis differentiated between PCR products such as primer-dimers and the desired product. Primer-dimers exhibited a lower Tm than the desired amplicon as they were considerably shorter (Brownie et al, 1997).…”

Identification of 8 Foodborne Pathogens by Multicolor Combinational Probe Coding Technology in a Single Real-Time PCR

“…However, such an observation does not compromise the overall performance of MCPC, because in the present setting pre-enriched samples provide much more DNA template than needed for each analysis. The problem may be partially solved by using single-tail strategy (26 ) to eliminate primer dimer formation and/or by extensive optimization of reaction conditions to reduce nonspecific amplification. Modified molecular beacons were used as probes in the present work because of their convenient availability.…”

Application of Multiplex PCR for Detection of Duchunne Muscular Dystrophy: A Childhood Neuromuscular Disorder

“…M-PCR also have some drawbacks which hinders during optimization such as poor sensitivity or specificity and/or preferential amplification of certain specific targets depending on different cases. The m-PCR utilizes more than one primer sets and at times it gives spurious amplification products other than the desired target due to primer dimers formation [57,58]. Therefore, the scientist's primarily focussing to minimize nonspecific interactions during optimization of m-PCR [59].…”