Sachadyn P, Kur J
Department of Microbiology, Technical University of Gdańsk, ul. Narutowicza 11/12, 80-952 Gdańsk, Poland.
Mol Cell Probes. 1998 Oct;12(5):259-62. doi: 10.1006/mcpr.1998.0170.
An example of the application and contruction of a polymerase chain reaction (PCR) internal control is presented. The internal control is synthesized in one PCR reaction. The primers used in this reaction possess 5' over-hanging ends which are identical to the primers used in the diagnostic reaction, whereas their 3' ends are complementary to a predetermined DNA sequence (pUC19 in this case) of defined length and sequence. As the sequence of the control except for primer sites, is not homologous to the PCR signal product, the formation of heteroduplexes and non-specific PCR products should not occur. Neither is there a risk that the target DNA will contaminate the internal control. However, the simultaneous amplification of two different DNA fragments flanked by the same primer sites resulted in either inhibition or enhancement of one or both products depending on the molar ratio of those DNA fragments. The presented method may be applied to construction of internal controls for quantitative PCR. The internal control was developed and tested for use in a PCR detection system for Agrobacterium tumefaciens.
本文展示了聚合酶链反应（PCR）内部对照的应用与构建实例。内部对照在一个PCR反应中合成。此反应中使用的引物具有5'突出末端，与诊断反应中使用的引物相同，而其3'末端与预定的特定长度和序列的DNA序列（本例中为pUC19）互补。由于除引物位点外，对照的序列与PCR信号产物不同源，因此不应形成异源双链体和非特异性PCR产物。目标DNA也不会污染内部对照。然而，由相同引物位点侧翼的两个不同DNA片段的同时扩增，根据这些DNA片段的摩尔比，会导致其中一种或两种产物受到抑制或增强。所提出的方法可应用于定量PCR内部对照的构建。该内部对照已开发并在用于根癌土壤杆菌的PCR检测系统中进行了测试。
