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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

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Author(s): Ponchel Frederique | Toomes Carmel | Bransfield Kieran | Leong Fong | Douglas Susan | Field Sarah | Bell Sandra | Combaret Valerie | Puisieux Alain | Mighell Alan | Robinson Philip | Inglehearn Chris | Isaacs John | Markham Alex

Journal: BMC Biotechnology
ISSN 1472-6750

Volume: 3;
Issue: 1;
Start page: 18;
Date: 2003;
Original page

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

ABSTRACT
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.
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