Protocol abstract
Nature Protocols 2, - 50 - 58 (2007)
Published online: 22 February 2007 | doi:10.1038/nprot.2007.11
Subject Categories: Genetic analysis | Nucleic acid based molecular biology
Anti-primer quenching-based real-time PCR for simplex or multiplex DNA quantification and single-nucleotide polymorphism genotyping
Jin Li1 & G Mike Makrigiorgos1
Abstract
Nucleic acid amplification and detection plays an increasingly important role in genetic analysis of clinical samples, medical diagnostics and drug discovery. We present a new quantitative PCR method that allows versatile and flexible nucleic acid target quantification. One of the PCR primers is modified by an oligonucleotide "tail" fluorescently labeled at the 5' end. An oligonucleotide complementary to this tail, carrying a 3'-quencher ("anti-primer"), is included in the PCR along with the two primers. Following primer extension, the reaction temperature is lowered such that the anti-primer hybridizes to and quenches the fluorescence of only the free primer and not the double-stranded PCR product, allowing real-time fluorescent quantification of the latter. This anti-primer-based quantitative real-time PCR (aQRT-PCR) allows simplex or multiplex quantification or single-nucleotide polymorphism genotyping in clinical samples of widely differing quality (e.g., fresh samples, formalin-fixed paraffin-embedded samples and plasma-circulating DNA) and provides a practical alternative to existing, more expensive approaches. The process of aQRT-PCR takes 1.5–2 h.
- Department of Radiation Oncology, Dana Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts, USA.
Correspondence to: G Mike Makrigiorgos1 e-mail: mmakrigiorgos@partners.org
nature-products
A-Z product listing
- 6-FAM- or ROX-labeled primers(Intergrated DNA Technologies)
- Absolute ethanol(Sigma)
- AmpliTaq DNA polymerase, Stoffel Fragment(Applied Biosystems)
- BHQ2-labeled anti-primer(Intergrated DNA Technologies)
- Dimethyl sulfoxide(Sigma)
- Distilled water(Invitrogen)