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 Biotechnol. 2003 Oct 13;3:18. doi: 10.1186/1472-6750-3-18.

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.

MeSH terms

  • Cell Line, Tumor
  • DNA / chemistry
  • DNA / genetics*
  • Female
  • Fluorescence
  • GTP Phosphohydrolases / genetics
  • Gene Amplification / genetics
  • Gene Deletion
  • Gene Rearrangement / genetics
  • Humans
  • Male
  • Oncogene Proteins / genetics
  • Optic Atrophy / genetics
  • Organic Chemicals / chemistry*
  • Polymerase Chain Reaction / methods*
  • Receptors, Antigen, T-Cell / genetics
  • Reproducibility of Results
  • Sensitivity and Specificity
  • Trans-Activators
  • Transcription Factors / genetics
  • Zinc Finger Protein GLI1

Substances

  • Oncogene Proteins
  • Organic Chemicals
  • Receptors, Antigen, T-Cell
  • Trans-Activators
  • Transcription Factors
  • Zinc Finger Protein GLI1
  • SYBR Green I
  • DNA
  • GTP Phosphohydrolases
  • OPA1 protein, human