Frederique Ponchel , 1 , Carmel Toomes 1 , Kieran Bransfield 1 , Fong T Leong 1 , Susan H Douglas 1 , Sarah L Field 1 , Sandra M Bell 1 , Valerie Combaret 2 , Alain Puisieux 2 , Alan J Mighell 1 , Philip A Robinson 1 , Chris F Inglehearn 1 , John D Isaacs 1 , Alex F Markham 1
13 October 2003
Real-time PCR is increasingly being adopted for RNA quantification and genetic analysis. At present the most popular real-time PCR assay is based on the hybridisation of a dual-labelled probe to the PCR product, and the development of a signal by loss of fluorescence quenching as PCR degrades the probe. Though this so-called 'TaqMan' approach has proved easy to optimise in practice, the dual-labelled probes are relatively expensive.
We have designed a new assay based on SYBR-Green I binding that is quick, reliable, easily optimised and compares well with the published assay. Here we demonstrate its general applicability by measuring copy number in three different genetic contexts; the quantification of a gene rearrangement (T-cell receptor excision circles (TREC) in peripheral blood mononuclear cells); the detection and quantification of GLI, MYC-C and MYC-N gene amplification in cell lines and cancer biopsies; and detection of deletions in the OPA1 gene in dominant optic atrophy.