Format

Send to

Choose Destination
Clin Chem. 2006 Apr;52(4):624-33. Epub 2006 Feb 9.

Antiprimer quenching-based real-time PCR and its application to the analysis of clinical cancer samples.

Author information

1
Department of Radiation Oncology, Dana Farber Cancer Institute, Harvard Medical School, Boston, MA, USA.

Abstract

BACKGROUND:

Nucleic acid amplification plays an increasingly important role in genetic analysis of clinical samples, medical diagnostics, and drug discovery. We present a novel quantitative PCR technology that combines the advantages of existing methods and allows versatile and flexible nucleic acid target quantification in clinical samples of widely different origin and quality.

METHODS:

We modified one of the 2 PCR primers by use of an oligonucleotide "tail" fluorescently labeled at the 5' end. An oligonucleotide complementary to this tail, carrying a 3' quenching molecule (antiprimer), was included in the reaction along with 2 primers. After primer extension, the reaction temperature was lowered such that the antiprimer hybridizes and quenches the fluorescence of the free primer but not the fluorescence of the double-stranded PCR product. The latter provides real-time fluorescent product quantification. This antiprimer-based quantitative real-time PCR method (aQRT-PCR) was used to amplify and quantify minute amounts of input DNA for genes important to cancer.

RESULTS:

Simplex and multiplex aQRT-PCR demonstrated linear correlation (r(2) >0.995) down to a DNA input equivalent to 20 cells. Multiplex aQRT-PCR reliably identified the HER-2 gene in microdissected breast cancer samples; in formalin-fixed, paraffin-embedded specimens; and in plasma circulating DNA from cancer patients. Adaptation to multiplex single-nucleotide polymorphism detection via allele-specific aQRT-PCR allowed correct identification of apolipoprotein B polymorphisms in 51 of 51 human specimens.

CONCLUSION:

The simplicity, versatility, reliability, and low cost of aQRT-PCR make it suitable for genetic analysis of clinical specimens.

PMID:
16469859
DOI:
10.1373/clinchem.2005.063321
[Indexed for MEDLINE]
Free full text

Supplemental Content

Full text links

Icon for HighWire
Loading ...
Support Center