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Received on November 7, 2005
Accepted on January 13, 2006
Molecular Diagnostics and Genetics |
1 Department of Radiation Oncology, Dana Farber Cancer Institute, Harvard Medical School, Boston, MA
2 Department of Medical Oncology, Dana Farber Cancer Institute, Harvard Medical School, Boston, MA
* To whom correspondence should be addressed. 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.
The following articles in journals at HighWire Press have cited this article:
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  C. A. Milbury, J. Li, and G. M. Makrigiorgos PCR-Based Methods for the Enrichment of Minority Alleles and Mutations Clin. Chem., April 1, 2009; 55(4): 632 - 640. [Abstract] [Full Text] [PDF]
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J. Li, L. Wang, P. A. Janne, and G. M. Makrigiorgos Coamplification at Lower Denaturation Temperature-PCR Increases Mutation-Detection Selectivity of TaqMan-Based Real-Time PCR Clin. Chem., April 1, 2009; 55(4): 748 - 756. [Abstract] [Full Text] [PDF]
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A. Di Nicola, E. Ghezzi, F. Gillio, F. Zerilli, E. Shehi, D. Maritano, M. Panizzo, F. Bonelli, and D. Adlerstein Anchor-Based Fluorescent Amplicon Generation Assays (FLAG) for Real-Time Measurement of Human Cytomegalovirus, Epstein-Barr Virus, and Varicella-Zoster Virus Viral Loads Clin. Chem., November 1, 2008; 54(11): 1900 - 1907. [Abstract] [Full Text] [PDF]
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C. Bonanno, E. Shehi, D. Adlerstein, and G. M. Makrigiorgos MS-FLAG, a Novel Real-Time Signal Generation Method for Methylation-Specific PCR Clin. Chem., December 1, 2007; 53(12): 2119 - 2127. [Abstract] [Full Text] [PDF]
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 G. Amicarelli, E. Shehi, G. M. Makrigiorgos, and D. Adlerstein FLAG assay as a novel method for real-time signal generation during PCR: application to detection and genotyping of KRAS codon 12 mutations Nucleic Acids Res., October 11, 2007; (2007) gkm809v1. [Abstract] [Full Text] [PDF]
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 F. Wang, L. Wang, C. Briggs, E. Sicinska, S. M. Gaston, H. Mamon, M. H. Kulke, R. Zamponi, M. Loda, E. Maher, et al. DNA Degradation Test Predicts Success in Whole-Genome Amplification from Diverse Clinical Samples J. Mol. Diagn., September 1, 2007; 9(4): 441 - 451. [Abstract] [Full Text] [PDF]
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J. Decock How Accurate Is the Antiprimer Quenching-Based Real-Time PCR for Detection of Her2/neu in Clinical Cancer Samples? Clin. Chem., July 1, 2006; 52(7): 1438 - 1439. [Full Text] [PDF]
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