Simple PCR-based method for synthesis of molecular calibrators and controls.

Abstract

The use of molecular methods such as PCR to quantify nucleic acids has become widely established for both research and diagnostic applications. Increasingly, such methodologies recognize the need for inclusion of internal standards and controls. These are used to define the efficiencies of nucleic acid extraction, reverse transcription, amplification, and detection and to quantify the effects of nonspecific inhibitors. Examples include the “Qs” controls included in Amplicor PCR assays (Roche Molecular Systems Inc.) (1) and the “Qa, Qb, Qc” controls used in Nuclisens assays (Organon Technica) (2). Control constructs (RNA or DNA) are derived from the nucleic acid sequence that the test method is designed to assay and span the primer binding sequences used in the assay. Thus, when added to the test sample, the control is coamplified in the reaction with a single primer pair. To distinguish the control from the test sequence during the detection phase of the assay, a short sequence between the primer binding sites is replaced by a defined sequence to allow differential probing with labeled oligonucleotides. Nucleic acid controls are usually 1 kb or longer, as shorter lengths may not by efficiently purified by the commonly used silica capture or precipitation methods. Previously, the construction of such controls required the identification or engineering of unique restriction sites between the primer binding sites to allow excision of the wild-type sequence and replacement with the probe sequence (1)(2). The process also required ligation of the probe sequence into the target sequence backbone and cloning into a plasmid containing a transcription promoter in the case of RNA controls. The method described here simplifies the procedure to a two-stage PCR amplification and allows the probe sequence to be placed anywhere between the primer binding sites without the need to identify or engineer restriction sites. Similar approaches …