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 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.
Brownie
J
J
Zeneca Diagnostics, Gadbrook Park, Northwich, Cheshire CW9 7RA, UK.
Shawcross
S
S
Theaker
J
J
Whitcombe
D
D
Ferrie
R
R
Newton
C
C
Little
S
S
D001483:Base Sequence; D017931:DNA Primers / Q000737:chemistry*; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction / Q000379:methods*
D017931:DNA Primers