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


CONCLUSIONS
The simplicity, versatility, reliability, and low cost of aQRT-PCR make it suitable for genetic analysis of clinical specimens.
Department of Radiation Oncology, Dana Farber Cancer Institute, Harvard Medical School, Boston, MA, USA.
Jin
J
Li
Fengfei
F
Wang
Harvey
H
Mamon
Matthew H
MH
Kulke
Lyndsay
L
Harris
Elizabeth
E
Maher
Lilin
L
Wang
G Mike
GM
Makrigiorgos
D001055:Apolipoproteins B / Q000235:genetics; D001943:Breast Neoplasms / Q000235:genetics / Q000473:pathology; D003110:Colonic Neoplasms / Q000235:genetics / Q000473:pathology; D004247:DNA / Q000097:blood; D017931:DNA Primers / Q000737:chemistry*; D005260:Female; D005404:Fixatives; D005456:Fluorescent Dyes / Q000737:chemistry*; D005557:Formaldehyde; D005838:Genotype; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics / Q000473:pathology; D009369:Neoplasms / Q000235:genetics* / Q000473:pathology; D009841:Oligonucleotides / Q000737:chemistry*; D010051:Ovarian Neoplasms / Q000235:genetics / Q000473:pathology; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction / Q000379:methods*; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000235:genetics; D015203:Reproducibility of Results
D001055:Apolipoproteins B; D004247:DNA; D017931:DNA Primers; D005404:Fixatives; D005456:Fluorescent Dyes; D005557:Formaldehyde; D009841:Oligonucleotides; D018719:Erb-b2 Receptor Tyrosine Kinases