Open Access Highly Accessed Methodology article

Real-time PCR based on SYBR-Green I fluorescence: An alternative to the TaqMan assay for a relative quantification of gene rearrangements, gene amplifications and micro gene deletions

Frederique Ponchel1*, Carmel Toomes1, Kieran Bransfield1, Fong T Leong1, Susan H Douglas1, Sarah L Field1, Sandra M Bell1, Valerie Combaret2, Alain Puisieux2, Alan J Mighell1, Philip A Robinson1, Chris F Inglehearn1, John D Isaacs1 and Alex F Markham1

Author Affiliations

1 Molecular Medicine Unit, University of Leeds, Leeds, UK

2 Centre Anti-cancer Leon Berard, Lyon, France

For all author emails, please log on.

BMC Biotechnology 2003, 3:18  doi:10.1186/1472-6750-3-18

Published: 13 October 2003

Abstract

Background

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.

Results

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.

Conclusion

Our assay has important clinical applications, providing accurate diagnostic results in less time, from less biopsy material and at less cost than assays currently employed such as FISH or Southern blotting.

Keywords:
Real-time PCR; SYBR-green; rearrangement; amplification; deletion