Strategy for controlling preferential amplification and avoiding false negatives in PCR typing.
Strategy for controlling preferential amplification and avoiding false negatives in PCR typing.
复制标题
PCR 分型中控制优先扩增并避免假阴性的策略。
作者：
T. Weissensteiner;J. Lanchbury
近年来，PCR方法在常规检测中的应用急剧增加。大多数分析涉及共同扩增的内部控制，几个等位基因在一个给定的位点或各种条带产生的低强度引物退火。在这种多重反应中，某些产物往往会优先放大。在次优PCR条件下，更敏感的扩增子可能会被淘汰，导致假阴性的分配。当扩增序列中稳定的双链DNA （dsDNA）区域引起假阴性时，优化PCR参数（如温度步骤、引物的相对浓度及其退火温度）并不能单独确保假阴性。本文提出了解决这一问题的两步策略：(i)用NaCl作为模型抑制剂滴定PCR，以建立发生假阴性的临界范围；（ii）在假阴性诱导条件下，用脱氧核糖核酸不稳定添加剂滴定PCR，直到调整共扩增片段的相对扩增效率。甜菜碱是一种新型高效的辅溶质。这些措施，以实现可靠的PCR分型一个困难的目标应该是有用的许多定性和定量多重PCR应用。
The use of the PCR method for routine testing has increased dramatically during recent years. Most assays involve co-amplification either of an internal control, of several alleles at a given locus or of a variety of bands produced by low-stringency primer annealing. In such multiplex reactions, certain products will often amplify preferentially. Amplimers that are more sensitive can be outcompeted under suboptimal PCR conditions, leading to assignment of false negatives. Optimization of PCR parameters such as temperature steps, relative concentrations of primers and their annealing temperature do not alone ensure against false negatives when caused by stable double-stranded DNA (dsDNA) regions in the amplified sequence. A two-step strategy to solve this problem is presented in this paper: (i) titration of the PCR with NaCl as a model inhibitor to establish the critical range within which false negatives occur; (ii) titration of the PCR with a dsDNA-destabilizing additive under false-negative-inducing conditions until the relative amplification efficiencies of co-amplified fragments are adjusted. Betaine is introduced as a novel and efficient cosolute. These measures to achieve reliable PCR typing of a difficult target should be useful for many qualitative and quantitative multiplex PCR applications.
影响因子：
2.9
作者：
REES, WA;YAGER, TD;VONHIPPEL, PH
通讯作者：
VONHIPPEL, PH