The elimination of primer-dimer accumulation in PCR
The elimination of primer-dimer accumulation in PCR
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DOI：
10.1093/nar/25.16.3235
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发表时间：
1997-08-15
影响因子：
14.9
通讯作者：
Little, S
中科院分区：
文献类型：
--
作者：
Brownie, J;Shawcross, S;Little, S
我们尝试从具有不同类型和互补程度的各种引物产生引物二聚体（PD），在产生PD的情况下，它们被克隆和测序，我们不能单独使用单个引物或使用相似序列引物产生可检测的PD，即使它们具有3'互补性。这些观察结果导致了这样的假设，即可以开发一种系统，由此可以消除PCR中PD的积累。我们证明了一种用于一般抑制PD形成的方法，该方法在扩增物的5'端使用一系列额外的核苷酸（尾）。加尾扩增物以低浓度存在，并且仅在PCR的早期循环期间参与。在随后的PCR循环中，使用具有与早期循环引物的尾部分相同的序列的单个引物实现扩增，这里我们将其称为标签。当产物小时，如PD，存在高局部浓度的源自尾的互补序列，这有利于由加尾引物相互作用产生的单链的互补末端的退火，并产生“泛柄”结构。这些的形成胜过另外的标签引物的退火，从而防止非特异性PD产物的积累。这有助于设计大型多重反应，并提供了一种通过使用嵌入染料直接在反应容器中检测特异性扩增子的方法。
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.