High primer concentration improves PCR amplification from random pools
High primer concentration improves PCR amplification from random pools
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DOI：
10.1093/nar/24.5.985
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发表时间：
1996-03-01
影响因子：
14.9
通讯作者：
Czerny, T
中科院分区：
文献类型：
--
作者：
Czerny, T
PCR的成功部分基于其指数扩增特性。然而，在实践中，可实现的产量是有限的。图1A和B显示了用不同但确定量的起始模板对典型反应的定量。每5个循环，取出反应的等分试样，然后定量累积产物的量。尽管最初所有反应的扩增速率在5个循环期间达到32倍扩增的理论极限（由虚线指示），但在随后的循环中观察到扩增速率急剧下降。有趣的是，在所有反应中发生的这种下降并不取决于每个反应中的循环总数，而是似乎与累积产物的量相关联。为了进一步了解这一现象的基础，我研究了引物浓度对PCR的影响。对于标准应用，建议引物浓度在0.1至1 µM之间（1），并且在反应过程中引物很少完全用完。然而，引物必须与积累的产物竞争以找到它们的靶序列，这可能在后期循环中限制反应。为了测试引物和产物浓度之间的比率是否影响产物产率，我用增加量的引物但在其他方面相同的条件下进行反应。对于测试的所有反应，观察到产物产率在后期而不是早期扩增轮次增加。尽管如此，
The success of PCR is partly based on its exponential amplification characteristics. Nevertheless, in practice, the achievable yields are limited. Figure 1A and B shows the quantification of a typical reaction with varying but defined amounts of starting template. Every five cycles an aliquot of the reaction was removed and afterwards the amount of accumulated product quantitated. Although, initially, the amplification rates of all reactions reached the theoretical limit of 32-fold amplification during five cycles (indicated by a dashed line), a sharp decline in amplification rates was observed at later cycles. Interestingly this decline, which occurred in all reactions, did not depend on the total number of cycles in each reaction, but rather seemed to be coupled to the amount of accumulated product. In order to learn more about the basis of this phenomenon, I investigated the effect of primer concentration on PCR. For standard applications a primer concentration between 0.1 and 1 µM is recommended (1), and rarely the primers are completely used up during the reaction. Nevertheless, the primers have to compete with the accumulating product in finding their target sequence, which could become limiting for the reaction at late cycles. To test whether the ratio between primer and product concentration influences the product yield, I performed reactions with increasing amounts of primers but otherwise identical conditions. For all reactions tested, an increase in product yield at late but not early rounds of amplification was observed. Nevertheless, the level of improvement strongly