Ponchel, F., Toomes, C., Bransfield, K., Leong, F.T., Douglas, S.H., Field, S.L., Bell, S.M., Combaret, V., Puisieux, A., Mighell, A.J., Robinson, P.A., Inglehearn, C.F., Isaacs, J.D. and Markham, A.F. (2003) 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. BMC Biotechnology, 3 (18). ISSN 1472-6750Full text available as:
Available under licence : See the attached licence file.
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.
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.
|Copyright, Publisher and Additional Information:||© 2003 Ponchel et al; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL.|
|Keywords:||Real-time PCR, SYBR-green, rearrangement, amplification, deletion|
|Institution:||The University of Leeds|
|Academic Units:||The University of Leeds > Faculty of Medicine and Health (Leeds) > Institute of Molecular Medicine (LIMM) (Leeds) > Section of Genetics (Leeds)|
|Depositing User:||Repository Officer|
|Date Deposited:||13 Mar 2006|
|Last Modified:||06 Jun 2014 07:59|