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
<p>Abstract</p> <p>Background</p> <p>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 o...
| Main Authors: | , , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2003-10-01
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| Series: | BMC Biotechnology |
| Subjects: | |
| Online Access: | http://www.biomedcentral.com/1472-6750/3/18 |
| _version_ | 1811271291517272064 |
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| author | Puisieux Alain Combaret Valerie Bell Sandra M Field Sarah L Douglas Susan H Leong Fong T Bransfield Kieran Toomes Carmel Ponchel Frederique Mighell Alan J Robinson Philip A Inglehearn Chris F Isaacs John D Markham Alex F |
| author_facet | Puisieux Alain Combaret Valerie Bell Sandra M Field Sarah L Douglas Susan H Leong Fong T Bransfield Kieran Toomes Carmel Ponchel Frederique Mighell Alan J Robinson Philip A Inglehearn Chris F Isaacs John D Markham Alex F |
| author_sort | Puisieux Alain |
| collection | DOAJ |
| description | <p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>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 <it>GLI</it>, <it>MYC</it>-C and <it>MYC</it>-N gene amplification in cell lines and cancer biopsies; and detection of deletions in the <it>OPA1 </it>gene in dominant optic atrophy.</p> <p>Conclusion</p> <p>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.</p> |
| first_indexed | 2024-04-12T22:17:23Z |
| format | Article |
| id | doaj.art-5ff7169c2bca41caa67ab684a7ae80f9 |
| institution | Directory Open Access Journal |
| issn | 1472-6750 |
| language | English |
| last_indexed | 2024-04-12T22:17:23Z |
| publishDate | 2003-10-01 |
| publisher | BMC |
| record_format | Article |
| series | BMC Biotechnology |
| spelling | doaj.art-5ff7169c2bca41caa67ab684a7ae80f92022-12-22T03:14:28ZengBMCBMC Biotechnology1472-67502003-10-01311810.1186/1472-6750-3-18Real-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 deletionsPuisieux AlainCombaret ValerieBell Sandra MField Sarah LDouglas Susan HLeong Fong TBransfield KieranToomes CarmelPonchel FrederiqueMighell Alan JRobinson Philip AInglehearn Chris FIsaacs John DMarkham Alex F<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>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 <it>GLI</it>, <it>MYC</it>-C and <it>MYC</it>-N gene amplification in cell lines and cancer biopsies; and detection of deletions in the <it>OPA1 </it>gene in dominant optic atrophy.</p> <p>Conclusion</p> <p>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.</p>http://www.biomedcentral.com/1472-6750/3/18Real-time PCRSYBR-greenrearrangementamplificationdeletion |
| spellingShingle | Puisieux Alain Combaret Valerie Bell Sandra M Field Sarah L Douglas Susan H Leong Fong T Bransfield Kieran Toomes Carmel Ponchel Frederique Mighell Alan J Robinson Philip A Inglehearn Chris F Isaacs John D Markham Alex F 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 Real-time PCR SYBR-green rearrangement amplification deletion |
| title | 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 |
| title_full | 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 |
| title_fullStr | 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 |
| title_full_unstemmed | 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 |
| title_short | 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 |
| title_sort | 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 |
| topic | Real-time PCR SYBR-green rearrangement amplification deletion |
| url | http://www.biomedcentral.com/1472-6750/3/18 |
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