Oligonucleotides used as template calibrators for general application in quantitative polymerase chain reaction

Abstract

The optimizing and controlling for polymerase chain reactions (PCRs) requires standard target sequences to measure reaction specificity and to obtain accurate gene quantification. However, defined target sequences are often not readily available. This situation is particularly evident in the study of rare splice variant transcripts. For gains in efficiency and reaction speed, a small size of PCR amplicon typifies real-time PCR formats, including hydrolysis probes. This study demonstrates the use of oligonucleotides resembling one strand of complete amplicon sequences used in real-time PCR to provide sustainable and precise amounts of the target sequence without the necessity of enlisting nucleic acid cloning procedures. The application of template oligonucleotides is modeled using all of the splice variant forms of human vascular endothelial growth factor.