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Clinical Chemistry 52: 624-633, 2006. First published February 9, 2006; 10.1373/clinchem.2005.063321
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(Clinical Chemistry. 2006;52:624-633.)
© 2006 American Association for Clinical Chemistry, Inc.

Molecular Diagnostics and Genetics

Antiprimer Quenching-Based Real-Time PCR and Its Application to the Analysis of Clinical Cancer Samples

Jin Li1, Fengfei Wang1, Harvey Mamon1, Matthew H. Kulke2, Lyndsay Harris2, Elizabeth Maher2, Lilin Wang1 and G. Mike Makrigiorgos1,a

Departments of1 Radiation Oncology and 2 Medical Oncology, Dana Farber Cancer Institute, Harvard Medical School, Boston, MA.

aAddress correspondence to this author at: Dana Farber–Brigham and Women’s Cancer Center, Brigham and Women’s Hospital, Level L2, Radiation Therapy, 75 Francis St., Boston, MA 02115. Fax 617-587-6037; e-mail mmakrigiorgos{at}partners.org.

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 (r2 >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.




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