Abstract
BackgroundReal-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.ResultsWe 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.ConclusionOur 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.
Highlights
Real-time PCR is increasingly being adopted for RNA quantification and genetic analysis
During the initial PCR cycles, the fluorescence signal emitted by SYBR-Green I bound to PCR product was usually too weak to register above background
We performed a quantification of T-cell receptor excision circles (TRECs) by two methods. (A) Samples were quantified against a standard curve run in parallel with the samples, to obtain an absolute quantity of TRECs per μg of DNA
Summary
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 duallabelled 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. The most popular, the 5'-nuclease assay, is based on the specific hybridisation of a duallabelled TaqMan [4] probe to the PCR product Another approach is based upon the binding of the fluorescent dye SYBR-Green I into the PCR product (PE Applied Biosystems, Warrington, UK). We report the development and rigorous testing of a real-time PCR assay using the inexpensive SYBRGreen I technology
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