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.
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.


CONCLUSIONS
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.
Ponchel
Frederique
F
Molecular Medicine Unit, University of Leeds, Leeds, UK. mmefp@leeds.ac.uk
Toomes
Carmel
C
Bransfield
Kieran
K
Leong
Fong T
FT
Douglas
Susan H
SH
Field
Sarah L
SL
Bell
Sandra M
SM
Combaret
Valerie
V
Puisieux
Alain
A
Mighell
Alan J
AJ
Robinson
Philip A
PA
Inglehearn
Chris F
CF
Isaacs
John D
JD
Markham
Alex F
AF
D052160:Benzothiazoles; D045744:Cell Line, Tumor; D004247:DNA / Q000737:chemistry / Q000235:genetics*; D003959:Diamines; D005260:Female; D005453:Fluorescence; D020558:GTP Phosphohydrolases / Q000235:genetics; D005784:Gene Amplification / Q000235:genetics; D017353:Gene Deletion; D015321:Gene Rearrangement / Q000235:genetics; D006801:Humans; D008297:Male; D015513:Oncogene Proteins / Q000235:genetics; D009896:Optic Atrophy / Q000235:genetics; D009930:Organic Chemicals / Q000737:chemistry*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011804:Quinolines; D011948:Receptors, Antigen, T-Cell / Q000235:genetics; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D015534:Trans-Activators; D014157:Transcription Factors / Q000235:genetics; D000071676:Zinc Finger Protein GLI1
D052160:Benzothiazoles; D003959:Diamines; D015513:Oncogene Proteins; D009930:Organic Chemicals; D011804:Quinolines; D011948:Receptors, Antigen, T-Cell; D015534:Trans-Activators; D014157:Transcription Factors; D000071676:Zinc Finger Protein GLI1; C098022:SYBR Green I; D004247:DNA; D020558:GTP Phosphohydrolases; C415198:OPA1 protein, human