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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.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Methods to detect and quantify nucleic acids are playing an increasing role in genetic analysis of clinical samples, medical diagnostics, and drug discovery; thus, the development of reliable, fast, and inexpensive DNA/RNA detection methods is important. Most methods use nucleic acid amplification via PCR (1) to enable detection and quantification of small amounts of target DNA/RNA from complex genomic DNA samples (2). A major advance for PCR-based nucleic acid detection has been the development of homogeneous, closed-tube assays using fluorescence detection that facilitate high-throughput detection and minimize the likelihood of false-positive results attributable to carryover contamination (3)(4)(5)(6)(7)(8)(9)(10)(11). By monitoring the PCR product at early stages of amplification, these "real-time" PCR technologies avoid inconsistencies associated with PCR saturation effects in the late stages of the reaction and enable target quantification in clinical samples with high reproducibility (3).

Real-time PCR methods can be divided into those using a hybridization probe (3)(4)(5)(9) and those using labeled PCR primers (6)(7)(8)(10)(11). The former provide high specificity and sensitivity in signal detection; however, the requirement for a third oligonucleotide other than the primers in the PCR reaction limits their multiplexing capability because of primer–probe interactions. As a result, separate PCR reactions for each genetic target are usually conducted, with accompanying increases in cost and labor. Approaches using universally labeled PCR primers can be more cost-effective as long as specificity does not become an issue because of nonspecific amplification or the formation of primer–dimers. Optimal primer design is therefore required in the latter case.

We present a novel method for signal generation in real-time PCR that uses fluorescently labeled PCR primers in combination with a universal quenching oligonucleotide (antiprimer) and combines many of the advantages of currently used approaches. The principle of multiplex antiprimer-based quantitative real-time PCR (aQRT-PCR) 1 is illustrated in Fig. 1 (the primers and antiprimers used in aQRT-PCR are depicted in more detail in Fig. 1 of the Data Supplement that accompanies the online version of this article at http://www.clinchem.org/content/vol52/issue4). One or more sets of primers are designed such that they carry a universal, 5'-positioned oligonucleotide "tail" fluorescently labeled with a different fluorophore for each target gene. An antiprimer carrying a molecule [3'-black hole quencher-2 (3'-BHQ2)] that quenches all fluorescent probes simultaneously is also included in the PCR reaction. The antiprimer has a lower melting temperature (Tm) than the primer and is complementary to the primer tail. PCR product is synthesized over the primer tail after the second PCR cycle (Fig. 1 ), yielding 5'-end-labeled fluorescent double-stranded DNA products. After polymerase synthesis at a temperature suitable for the primer Tm, the reaction temperature is lowered by 5–10 °C to allow the antiprimer to bind and quench the free primer, and the fluorescence of the PCR product is then recorded. The polymerase synthesis step thus is decoupled from the signal detection step. Because the quenching molecule can be placed at any position along the antiprimer, the present approach allows the flexibility to achieve ideal quenching of the fluorescent primer, which enables strong signal generation. Below we demonstrate that this new real-time PCR methodology offers high sensitivity, reproducibility, and multiplexing capability for detecting both gene amplifications and single-nucleotide polymorphisms (SNPs). The cost for aQRT-PCR is lower than for most current approaches, and this approach offers several advantages in the analysis of clinical samples. In validating the new method, we also developed a multiplex, single-tube methodology, using aQRT-PCR, to detect HER-2 copy number variations in microdissected breast cancer samples; in formalin-fixed, paraffin-embedded (FFPE) specimens; and in plasma circulating DNA.


Figure 1
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  		Figure 1. Outline of aQRT-PCR.

After the initial PCR cycling with fluorescently labeled primers, the temperature of the reaction is decreased to allow a quencher-labeled oligonucleotide (antiprimer) to bind the unincorporated primer. The antiprimer cannot bind to the double-stranded PCR product, which generates an exponentially increasing signal. Signal can be simplex or multiplex (also see Fig. 1 in the online Data Supplement).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
source and extraction of genomic dna
Reference human male genomic DNA was purchased from Promega. BT474 genomic DNA was purified from a cultured BT474 breast cancer cell line obtained from the American Tissue Culture Collection. The 51 human surgical lung tissue samples were obtained from the Massachusetts General Hospital Tumor Bank, and the 4 Her2+ breast cancer samples were obtained from the Dana Farber Cancer Center SPORE Bank after manual microdissection. The FFPE specimens were obtained from the Department of Pathology, Brigham and Women’s Hospital. Plasma samples were obtained from the Medical Oncology Tumor Bank, Dana Farber Cancer Institute. The use of unidentifiable human specimens for genetic analysis was approved by the Institutional Review Board. Genomic DNA from the BT474 cell line and from fresh tissues was extracted and purified by use of the DNAeasy KitTM (Qiagen). A modified method was used for extracting DNA from FFPE specimens. Briefly, 25 mg of tissue per sample was deparaffinized by treatment with mixed xylenes. Deparaffinization involved mixing the sample with 1.2 mL of xylenes, vortex-mixing, centrifuging the mixture for 3 min at room temperature, and removing the xylene layer; this procedure was repeated 1 or 2 times until the xylene layer was clear. After deparaffinization, the xylenes were removed by the addition of absolute ethanol (1.2 mL), vortex-mixing, centrifugation for 3 min at room temperature, and removal of the ethanol layer; this procedure was repeated 1–2 times until the ethanol layer was clear. After vaporization of the ethanol by incubation for 10 min at 37 °C, samples were washed in phosphate-buffered saline (addition of 1.2 mL of phosphate-buffered saline, vortex-mixing, centrifugation for 3 min at room temperature). After removal of the phosphate-buffered saline, the tissue was placed in 360 µL of lysis buffer (Qiagen) + 40 µL of proteinase K (concentrated) and rotated at 55 °C for 24–72 h as needed for full digestion. Subsequent DNA purification was carried out with the DNAeasy Kit, adjusting buffer and extraction volumes for the volume of lysis buffer used. The quality of the extracted DNA was initially evaluated by gel electrophoresis of 0.75 µg of DNA in a 1% agarose gel. To extract plasma circulating DNA, within 2–5 h of collection, whole blood was centrifuged at 2000g for 15–30 min, and the plasma was carefully collected from the top of the supernatant, as described previously (12). Plasma circulating DNA was purified from plasma with QIAampTM MinElute Virus Spin Kit (Qiagen) and quantified by the PicoGreenTM method (Molecular Probes).

single-gene (simplex) AQRT-PCR
For simplex aQRT-PCR, amplification was performed with the AmpliTaq GoldTM amplification reagents (Applied Biosystems) in a Smart-CyclerTM real-time thermocycler (Cepheid). The fluorescently labeled primer and nonlabeled primers (Table 1 ) were designed with Oligo 6 software (Molecular Biology Insights) and synthesized at Integrated DNA Technologies. 6-Carboxyfluorescein (FAM) and 6-carboxy-X-rhodamine (ROX) N-hydroxysuccinimide ester were used as labels at the 5' end of the forward or, alternatively, the reverse fluorescence primer. Serial dilutions of DNA (0.14–145 ng) in a 1-µL volume were added to a final volume of 20 µL with a final concentration of 1x ABI TaqMan master mixture (Applied Biosystems), 0.2 µM each fluorescently labeled primer, unlabeled primer, and 1 µM BHQ2-labeled antiprimer (synthesized at Integrated DNA Technologies; Table 1 ). The thermocycling program was 1 cycle of 50 °C for 2 min, 1 cycle of 95 °C for 10 min, and 40–50 cycles of 95 °C for 15 s, 60 °C for 30 s, 50 °C for 30 s, and 50 °C for 15 s (for reading fluorescence).


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  		Table 1. Primers and probes for aQRT-PCR.

multiplex AQRT-PCR and determination of HER-2 gene amplification
Multiplex aQRT-PCR was performed to simultaneously detect HER-2 (FAM channel) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; ROX channel) in a single tube. Serial dilutions of DNA (0.14–145 ng) in a 1-µL volume from reference or BT474 cells was added to a final volume of 20 µL containing a final concentration of 1x ABI TaqMan master mixture, 0.05 µM each FAM-labeled HER-2 reverse primer and unlabeled HER-2 forward primer, 0.15 µM each ROX-labeled GAPDH forward primer and unlabeled GAPDH reverse primer, and 1 µM BHQ2-labeled antiprimer. The thermocycling program was 1 cycle of 50 °C for 2 min, 1 cycle of 95 °C for 10 min, and 40 cycles of 95 °C for 15 s, 60 °C for 30 s, 50 °C for 30 s, and 50 °C for 15 s (for reading fluorescence). Fluorescence was read in both the FAM and ROX channels simultaneously. Three independent experiments were performed for each gene to generate a mean (SD) relative copy number. The relative gene amplification ratio between unamplified/amplified plasma circulating DNA was calculated by the comparative threshold cycle ({Delta}{Delta}Ct) method (3)(13).

To examine clinical samples for HER-2 amplification by multiplex aQRT-PCR, we used 2 ng of genomic DNA from the microdissected breast cancer samples (Her2+) in the reaction. For the FFPE samples, we used 20 ng as input genomic DNA, and for the plasma circulating DNA samples, we used 1 µL from each Qiagen-purified DNA sample in the reaction.

TAQMAN real-time pcr
We also performed TaqMan real-time PCR for HER-2 to compare with the results obtained by aQRT-PCR. The assay was done as described previously (13)(14). Briefly, amplification was performed with AmpliTaq Gold (Applied Biosystems) in a Smart-Cycler. Primers and probes for exonic regions of genes studied were synthesized by Biosearch Technologies. For each reaction, we added 2 ng of genomic DNA to a final volume of 20 µL with final concentrations of 1x ABI TaqMan master mixture (Applied Biosciences), 0.2 µM each primer, and 0.1 µM probe. The thermocycling program was as follows: 1 cycle of 50 °C for 2 min, 1 cycle of 95 °C for 10 min, 50 cycles of 95 °C for 15 s and 60 °C for 1 min. Three independent experiments were performed for each gene to generate a mean (SD) relative copy number by the {Delta}{Delta}Ct method.

allele-specific AQRT-PCR of b71 snp
We adapted the published allele-specific PCR primers for the B71 SNP (C>T) of human apolipoprotein B (15) by adding different fluorescent probes to the tails of each allele. The ROX-labeled forward primer, specific for the C genotype (indicated by the italic font), was 5'-ROX-AGTGCTATCCGAGGGAAGAAGACCAGCCAGTGCAC-3', the FAM-labeled forward primer, specific for the T genotype, was 5'-FAM-AGTGCTATCCGAGGGAATGAAGACCAGCCAGTGCAT-3', and the reverse primer was 5'-CAAGGCTTTGCCCTCAGGGTT-3'. The PCR reaction was conducted in a 20-µL volume using a Smart-Cycler real-time PCR instrument. The real-time PCR reaction was set up as follows: 40 ng of human genomic DNA from clinical lung samples, 0.2 µM each C- or T-genotype–specific forward primer; 0.2 µM reverse primer; 1x Stoffel polymerase buffer; an extra 30 mM KCl to a final concentration of 40 mM; 2 mM MgCl2; 50 µM each of dATP, dCTP, dGTP, and dTTP; 50 nL/µL dimethyl sulfoxide; 25 nL/µL glycerol; and 2 U of Stoffel Taq polymerase (Perkin-Elmer). The thermocycling program was 1 cycle of 95 °C for 2 min and 40 cycles of 95 °C for 15 s, 60 °C for 30 s, 50 °C for 30 s, and 50 °C for 15 s (for simultaneous reading of fluorescence from the FAM and ROX channels). For multiplex genotyping, AmpliTaq Gold (Applied Biosystems) was used instead of the Stoffel Taq polymerase. To genotype the B71 SNP, we performed multiplex real-time PCR in a final volume of 20 µL with the following final concentrations: 1x ABI TaqMan master mixture (Applied Biosystems), 40 ng of genomic DNA, 0.05 µM FAM-labeled T-specific primer, 0.15 µM ROX-labeled C-specific primer, 0.2 µM unlabeled reverse primer, and 1 µM BHQ2-labeled antiprimer. The thermocycling program was 1 cycle of 50 °C for 2 min, 1 cycle of 95 °C for 10 min, and 40 cycles of 95 °C for 15 s, 60 °C for 30 s, 50 °C for 30 s, and 50 °C for 15 s (for reading fluorescence from the FAM and ROX channels). To determine the reproducibility of the multiplex aQRT-PCR approach for genotype determination, we repeated the experiments 10 separate times.

multiplex pcr for genotyping the b71 snp in clinical samples
We performed multiplex real-time PCR to genotype the apolipoprotein B (B71) SNP in 51 clinical lung samples, using the same PCR protocol with 40 ng of genomic DNA as starting material.

To verify the results of the aQRT-PCR multiplex genotyping, direct PCR sequencing on all 51 samples was performed by Polymorphic DNA Technologies, Inc. (Alameda, CA) and by the Dana Farber Molecular Biology Core Facility.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
design of fluorescently labeled primers and antiprimer
aQRT-PCR is designed to enable real-time PCR in simplex or multiplex formats for gene copy number determination and SNP genotyping using primers containing universal fluorescently labeled oligonucleotide tails (see Fig. 1 in the online Data Supplement). The tail consists of a 17-base oligonucleotide added to the 5' end of the forward or, alternatively, the reverse gene-specific primer, depending on which placement is less likely to contain secondary (hairpin) structures, as predicted by Oligo 6 software. In the current applications, the Tm of this oligonucleotide tail is ~57 °C, as calculated by Oligo 6 software. The antiprimer complementary to this tail also has a Tm of ~57 °C, whereas the gene-specific portion of the primer is designed to have a higher Tm (~65 °C). At an annealing-extension PCR temperature of 60 °C, the primers can bind the target and induce polymerization without much interference from the lower-Tm antiprimer. When the temperature is subsequently decreased to 50 °C, the antiprimer binds the tail of the free, single-stranded primer, but not the double-stranded PCR product (Fig. 1Up ). Because the antiprimer concentration is 2- to 4-fold higher than that of the primer concentration, the majority of the free primer is expected to bind the antiprimer at 50 °C, thus strongly quenching the primer fluorescence. Because the 5' end of the primer tail is opposite to the 3' end of the antiprimer, the interaction is mediated via exciton interaction (16)(17) (i.e., direct contact-quenching) between the 5'-fluorophore and the 3'-quencher present on the tail and antiprimer, respectively, which for most fluorophores provides stronger quenching than fluorescence resonance energy transfer (18). The quencher chosen here was BHQ2, which has a wide range of absorbance wavelengths; it thus is appropriate for quenching multiple fluorophores simultaneously, including the FAM and ROX used for multiplex PCR in the present work.

Careful design of primers by use of Oligo 6 minimizes the probability for secondary structures and primer–dimer formation in aQRT-PCR. Furthermore, by its design, the antiprimer does not participate in primer–dimer formation because the 3'-quenching molecule is also an effective polymerase block (4).

simplex AQRT-PCR
We used aQRT-PCR for simplex amplification and quantification of several genes from human genomic DNA (Table 1Up ). Shown in Fig. 2A are primary amplification curves obtained with various amounts of starting genomic reference DNA, down to an equivalent of 20 cells, for the gene HER-2, and the calibration curve (log concentration vs Ct) is shown in Fig. 2B . For comparison, the TaqMan real-time PCR method was performed in parallel (Fig. 2 , C and D). The 2 methods used the same PCR reagent set, annealing and extension temperatures, fluorophore and quencher, and essentially the same primers except that the forward primer used in aQRT-PCR also contains the 5'-FAM-labeled tail. Under essentially identical PCR conditions, aQRT-PCR generated significantly stronger fluorescence signals than did the TaqMan reaction (Fig. 2 ), which possibly reflects the stronger quenching effect produced by direct contact of FAM and BHQ2 in aQRT-PCR compared with the fluorescence energy transfer in TaqMan. The data also demonstrate that the 2 methods have similar Pearson correlation coefficients (r2), indicating their equivalency for simplex HER-2 quantification.


Figure 2
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  		Figure 2. Comparison of simplex aQRT-PCR with TaqMan-based real-time PCR for the HER-2 gene.

Serial dilutions of input reference genomic DNA (0.14–145 ng) were amplified with aQRT-PCR and TaqMan in parallel. (A), primary amplification curve for aQRT-PCR; (B), log concentration of input genomic DNA vs Ct for aQRT-PCR; (C), primary amplification curve for TaqMan assay; (D), log concentration of input genomic DNA vs Ct for TaqMan PCR.

We tested 4 additional genes (TBP, MYC, HBEGF, and TOP1), which have different amplicon sizes (69–134 bp), by simplex aQRT-PCR performed in triplicate independent experiments. Representative primary amplification curves are depicted in Fig. 2 in the online Data Supplement). The results demonstrate strong signals and linear curves for log concentration vs threshold (r2 >0.99), with a lower limit of input genomic DNA of ~20 cell equivalents (~0.1 ng of genomic DNA). The no-DNA controls (water) gave no detectable signals for at least 45–50 PCR cycles with the primers tested.

multiplex AQRT-PCR
We performed multiplex aQRT-PCR according to the scheme shown in Fig. 1Up , using FAM-labeled reverse and ROX-labeled forward primers for the HER-2 oncogene and the GAPDH housekeeping gene, respectively, to quantify HER-2 amplification in a single-tube reaction. Both fluorescently labeled primers have the same oligonucleotide tail as the simplex aQRT-PCR. For optimal coamplification of HER-2 and GAPDH, the ratio of FAM- and ROX-labeled primers was experimentally determined to be 1:3. Under these conditions, the 2 genes are amplified with similar amplification efficiencies when reference genomic DNA was used (Fig. 3A in the online Data Supplement). We next tested serial dilutions of the starting material, human male reference genomic DNA (0.14–145 ng); the multiplex aQRT-PCR response was linear on both channels simultaneously (panels B and C in Fig. 3 of the online Data Supplement). The multiplex assay was linear (r2 ~0.995) down to an amount of starting material equivalent to 20 cells, whereas the negative control (water) was negative in both the FAM and ROX channels for at least 45 PCR cycles.

The ability of the multiplex assay to quantify, in a single reaction, the known amplification of the oncogene HER-2 in genomic DNA from BT474 breast cancer cells is shown in panels D and E in Fig. 3 of the online Data Supplement. The GAPDH-normalized threshold difference ({Delta}{Delta}Ct = 4.2) is in good agreement to that obtained with the simplex TaqMan assay, in 2 separate reactions ({Delta}{Delta}Ct = 3.9), for BT474 genomic DNA, as per our earlier report (14). Furthermore, in triplicate repeated experiments, a 20% dilution of BT474 DNA in reference DNA was reliably differentiated from pure reference DNA (curves 3–5 in panels D and E of Fig. 3 in the online Data Supplement). This indicates that the method should be able to detect a 20% minority of HER-2 amplified cancer cells in a background of 80% stromal cells.

We next examined the {Delta}{Delta}Ct for BT474 cells when the starting genomic DNA material was gradually decreased from 145 ng down to 0.14 ng. The results (Fig. 3F in the online Data Supplement) indicated that the {Delta}{Delta}Ct remains substantially constant even at low input DNA (relative standard deviation of the {Delta}{Delta}Ct, 13%), indicating the ability of the multiplex approach to reliably quantify gene amplification in samples containing minute amounts of DNA, such as those obtained from fine-needle biopsies or from tissue microdissection.

HER-2 amplification in clinical samples detected by multiplex AQRT-PCR
HER-2 is overexpressed in 20%–30% of breast cancers (19), ovarian cancer(20), and other cancers (21)(22), and its expression is correlated with clinical outcome (19). To demonstrate the ability of multiplex aQRT-PCR to detect HER-2 amplification in fresh DNA from microdissected clinical samples, we tested HER-2 amplification in DNA extracted from 4 manually dissected breast cancer specimens characterized as HER2+ by immunohistochemistry and fluorescence in situ hybridization in our institution (19). Shown in panels A and B of Fig. 3 are the primary amplification curves obtained with 2 ng of starting DNA (equivalent to ~350 cells) for the 4 samples and for reference and BT474 DNA by multiplex aQRT-PCR for HER-2/GAPDH. All 4 samples gave substantial (>8-fold) chromosomal HER-2 amplification, in agreement with the fluorescence in situ hybridization and immunohistochemistry results. The threshold difference ({Delta}{Delta}Ct) for the 4 samples ranged between 3 and 5 cycles, similar to the amplification detected in BT474 breast cancer cells.


Figure 3
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  		Figure 3. Multiplex aQRT-PCR of clinical samples for HER-2 amplification.

(A and B), microdissected samples from patients with stage IA breast cancer, as measured in the GAPDH (A) and HER-2 (B) channels. Microdissected sample 334, {Delta}{Delta}Ct = 5.5; microdissected sample 438, {Delta}{Delta}Ct = 2.9; microdissected sample 637, {Delta}{Delta}Ct = 5.8; microdissected sample 408, {Delta}{Delta}Ct = 3.5; reference DNA, {Delta}{Delta}Ct = 0; BT474 DNA, {Delta}{Delta}Ct = 4.1. (C and D), DNA from FFPE specimens. FFPE 2, {Delta}{Delta}Ct = 0.7; FFPE 3, {Delta}{Delta}Ct = 1.4; FFPE 19, {Delta}{Delta}Ct = 1.0; FFPE 56, {Delta}{Delta}Ct = 0.1. (E and F), plasma circulating DNA from 4 blood samples. Plasma 4, obtained from a colon cancer patient, displays HER-2 amplification ({Delta}{Delta}Ct = 2.6), whereas for the other samples, no amplification is detectable.

To examine the utility of the method in situations in which the starting DNA material is of low quality and/or quantity, we applied multiplex aQRT-PCR to the detection of HER-2 in DNA from FFPE specimens as well as in free circulating DNA extracted from plasma of colon and ovarian cancer patients. DNA extracted from these clinical samples is highly fragmented and is often difficult to amplify (12)(14)(23). As shown in panels C and D of Fig. 3Up , multiplex HER-2/GAPDH amplification using DNA from FFPE samples (20 ng each) obtained from glioma cancer patients revealed that the formalin fixation procedure caused considerable DNA degradation (Fig. 4 in the online Data Supplement; agarose gel images). Compared with the threshold obtained for the reference DNA (10 ng of DNA) in the same experiment, aQRT-PCR was not significantly affected by the fragmentation in the starting material. Multiplex HER-2/GAPDH amplification results for 4 plasma circulating DNA samples obtained from colon and ovarian cancer patients are shown in panels E and F of Fig. 3Up . In this case, 1 µL of purified DNA was used in each reaction, and 2 independent experiments were performed. One of the plasma samples obtained from a colon cancer patient (sample 4) harbored an ~6-fold HER-2 amplification, whereas the remaining plasma samples were negative for amplification. The results indicate that for short amplicons, such as those used for HER-2 and GAPDH (~70 bp each), multiplex aQRT-PCR is not significantly affected by fragmentation of the input material.

real-time simplex and multiplex snp genotyping by AQRT-PCR
To adapt aQRT-PCR for real-time SNP genotyping, we used allele-specific PCR based on a 3'-mismatched nucleotide (24)(25). We selected a well-studied polymorphism of the apolipoprotein B gene (B71; C>T) for validation of the method. The T-allele–specific primer was labeled with FAM, and the C-allele–specific primer was labeled with ROX; the reverse primer was unlabeled and was shared by the allele-specific primers. A common antiprimer was used to quench both allele-specific primers. Two DNA samples previously sequenced at the Dana Farber Core sequencing facility and known to be homozygous C/C and T/T were first tested by simplex aQRT-PCR using the Stoffel fragment of Taq polymerase. We found an 8-cycle threshold difference between the 2 alleles, corresponding to an ~256-fold difference in the fluorescence signal (Fig. 4A ). We then applied the method in a multiplex format, using Stoffel Taq polymerase, but the reaction yield was suboptimal (data not shown). We therefore used multiplex aQRT-PCR with the AmpliTaq Gold polymerase. For multiplex aQRT-PCR, the ratio of FAM- and ROX-labeled allele-specific primers yielding optimal signals for both alleles in our real-time PCR instrument was experimentally determined to be 1:3. The multiplex SNP genotyping results for 3 genomic DNA samples (40 ng each) containing the C/C (homozygous), C/T (heterozygous), and TT (homozygous) genotypes are shown in Fig. 4B . The genotypes of these 3 samples were verified by sequencing (Fig. 5 in the online Data Supplement). To examine the reproducibility of the multiplex approach, we repeated the experiment 9 more times; the mean (SD) {Delta}Ct values are shown in Fig. 4C . The Ct values obtained differed by 4.5 (0.7) cycles (C/C) and 3.5 (0.4) cycle (T/T) between each of the 2 homozygous samples and the heterozygous (C/T) sample. Assuming Poisson statistics, the threshold range covered by ({Delta}Ct ± 3 SD) is expected to cover 99.73% of the distribution of values (26). Accordingly, multiplex aQRT-PCR was able to differentiate the 3 apolipoprotein B genotypes with high confidence.


Figure 4
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  		Figure 4. Simplex and multiplex aQRT PCR genotyping of the apolipoprotein B SNP B71.

(A), simplex allele-specific aQRT-PCR using Stoffel Taq polymerase. ROX channel represents the C genotype; FAM channel represents the T genotype. aQRT-PCR results obtained with genomic DNA from 2 specimens with known C/C or T/T genotypes are depicted. (B), multiplex aQRT-PCR genotyping using AmpliTaq Gold polymerase performed on DNA predicted to be the C/C, CT, or TT genotype. (C), results of 10 independent repeats of the multiplex experiment for the 3 genomic DNAs. The mean (SD; error bars) Ct values are shown. The genotypes of these 3 samples were verified by sequencing (see Fig. 5 in the online Data Supplement).

multiplex snp genotyping of clinical samples by AQRT-PCR
To further validate the use of multiplex aQRT-PCR in clinical samples, we used the method to determine the apolipoprotein B genotype in genomic DNA extracted from 51 surgical lung specimens. Duplicate independent multiplex aQRT-PCR experiments were carried out, and the mean {Delta}Ct (ROX-FAM) was calculated. We determined genotype by comparing the {Delta}Ct to the ({Delta}Ct ± 3 SD) range of genotype-specific thresholds derived from the experiment shown in Fig. 4CUp . The DNA was sequenced in parallel. The results of this study indicated complete (51 of 51) agreement between the 2 independent methods (Table 2 ).


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  		Table 2. Comparison of genotyping results obtained by multiplex aQRT-PCR and by sequencing for 51 clinical lung samples.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We validated aQRT-PCR, a real-time PCR methodology that is a novel modification of principles originally described by Nazarenko et al. (7), Morrison et al.(27), and Li et al. (28). The new method uses primers containing a universal oligonucleotide tail labeled with a single fluorescent reporter molecule. In multiplex PCR applications, primers containing different fluorescent probes were used in conjunction with a single probe (antiprimer) carrying a 3' quencher. aQRT-PCR thus could be used to quantify a wide range of targets in simplex or multiplex reactions in conjunction with one-time synthesis of the antiprimer. By decoupling the probe carrying the fluorophore from the probe carrying the quencher, the current approach substantially reduces the overall cost relative to real-time PCR methods requiring double labeling of every gene-specific primer or probe (3)(4)(5)(8)(9)(10)(11). As a result, aQRT-PCR lends itself to cost-effective adaptation for high-throughput applications.

In a comparison with the TaqMan assay for real-time quantification of the HER-2 gene, aQRT-PCR demonstrated similar sensitivity while providing stronger fluorescent signals attributable to the optimal (contact) quenching made possible between the fluorescent probe and quencher by the antiprimer design. Because the TaqMan assay uses an oligonucleotide probe that must bind the template for signal generation, the TaqMan assay is still expected, in principle, to provide higher target specificity than methods that use fluorescent primers, such as aQRT-PCR. On the other hand, signal generation in aQRT-PCR is not dependent on the 5'->3' exonucleolytic action of the polymerase, thereby enabling use of a variety of polymerases, including improved, high-processivity, low-cost polymerases that lack this property. In addition, similar to all methods that use a probe for signal generation, multiplexing using the TaqMan assay is difficult because of the increased probability for primer–probe interactions during PCR. Thus, 2 separate reactions must used for detection of HER-2 gene copy number changes in breast cancer samples (14). This may increase the uncertainty in target quantification for several reasons. For example, inherent factors that can lead to between-tube or between-sample variability are thermocycler-dependent temperature deviations, the presence of individual DNA polymerase inhibitors in clinical samples, pipetting variations, or the abundance of the target sequence in the specimen of interest (2).

In contrast, adaptation of aQRT-PCR to the multiplex quantification of clinically relevant targets was straightforward, as demonstrated by the ability of the assay to detect HER-2 oncogene amplification in clinical cancer specimens from diverse sources and input DNA quantities and qualities (microdissection, FFPE specimens, plasma). The single-tube format of multiplex aQRT-PCR makes this technique highly reproducible because between-sample variability is minimized. As a result, it is possible to reliably identify HER-2 amplification in the presence of up to 80% background DNA. Detection of HER-2 amplification in the presence of excess nontumor (stromal) cells may provide an alternative to the costly and laborious approach of characterizing surgical tissue specimens by fluorescent in situ hybridization (19).

Identification of SNPs by aQRT-PCR in clinical samples by use of allele-specific PCR is another useful adaptation of the technology that could find wide applications both in medicine and biotechnology. Detection of the apolipoprotein B B71 polymorphism in clinical samples in multiplex format was reliable and in agreement with sequencing in 51 of 51 cases (Table 2Up ). The ability to perform multiplex SNP detection in a single tube is expected to decrease inherent PCR uncertainties and minimize cost and labor, thus enabling the technology to potentially be used in SNP quantification in allele pools (29)(30), for rapid assessment of sample quality after DNA/RNA extraction from FFPE samples or bodily fluids (23), and in other applications.

In summary, we present a novel real-time PCR methodology, aQRT-PCR, which combines simplicity, versatility, and low cost. We demonstrated the applicability and reliability of the aQRT-PCR method to the analysis of clinical samples in diverse situations. We also demonstrated a new approach for the evaluation of HER-2 gene copy amplification in clinical cancer samples. We expect that aQRT-PCR will find a wide range of applications in medicine and biotechnology.


   Acknowledgments
 
We gratefully acknowledge the assistance of Mohamet Miri and Frank Haluska, MD, in obtaining tissue specimens from the Massachusetts General Hospital Tumor Bank. This work was supported by NCI Grants 1 R21 CA 111994-01 and CA 115439-01 and, in part, by a Technology Award from the Dana Farber-Harvard Cancer Center, by the JCRT Foundation, and by the Dana-Farber/Harvard Cancer Center SPORE in Breast Cancer. F.W. was supported by Training Grant 2 T32 CA 009078-31.


   Footnotes
 
1 Nonstandard abbreviations: aQRT-PCR, antiprimer-based quantitative real-time PCR; BHQ2, black hole quencher-2; Tm, melting temperature; SNP, single-nucleotide polymorphism; FFPE, formalin-fixed, paraffin-embedded (specimens); FAM, 6-carboxyfluorescein; ROX, 6-carboxy-X-rhodamine; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; and Ct, threshold cycle.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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