The construction and use of a PCR internal control

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. (C) 1998 Academic Press.