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Quantitative polymerase chain reaction-based homogeneous assay with fluorogenic probes to measure c-erbB-2 oncogene amplification

¹ Perkin-Elmer Italy, Monza, Milan, Italy.
^a Author for correspondence. Fax +39.55.4377290.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
We describe a PCR-based assay for determining c-erbB-2 oncogene amplification in breast cancer in which we use the TaqMan^TM system. Two fluorogenic probes anneal to the target between primers for c-erbB-2 and ß-globin genes and contain both a reporter dye (6-carboxy-fluorescein) and a quencher dye (6-carboxy-tetramethyl-rhodamine). During the extension phase of the PCR cycle, the 5'3' exonuclease activity of Taq polymerase cleaves the hybridized fluorogenic probe, resulting in an increase of fluorescence emission of the reporter dye that is quantitative for the amount of PCR product and, under appropriate conditions, for the amount of template. Assay performance showed adequate precision and a lower detection limit and good correlation with the results obtained in the same samples by a competitive PCR assay (n = 25, r = 0.94, P <0.01). This homogeneous assay is time-saving, avoids usually cumbersome postamplification procedures (that can be additional sources of inaccuracy and contamination), and seems suitable for determination of c-erbB-2 oncogene amplification in tumor specimens.

Key Words: indexing terms: breast cancer • genetic alterations • oligonucleotide probes • fluorescence assays

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Several processes for quantitative PCR have been proposed. These approaches have been addressed by either assessments of procedures for accurate measurement of PCR products (1)(2)(3)(4)(5) or strategies involving coamplification of an external reference gene (differential PCR) (6)(7) or an internal standard (competitive PCR) (8)(9)(10). Independently from the various strategies adopted, however, measurements of a nucleic acid target (DNA or mRNA) by PCR require time-consuming postamplification steps that can, to some extent, introduce additional uncontrolled variables and risks of carryover contamination. Thus, any evolution of PCR techniques that allows direct detection of specific amplified targets without any post-PCR processing is relevant.

Recently, detection of specific PCR products with fluorogenic probes has been proposed (11)(12)(13)(14). The probes are designed to hybridize within the target sequence and to generate a signal that accumulates during PCR cycling in proportion to the accumulation of amplification products. The end-point measurement of fluorescence in each sample thus provides a homogeneous signal that is specifically associated with the amplified target and quantitatively related to the amount of PCR products, without requiring further post-PCR procedures.

To verify the applicability of this approach to quantitative PCR, we developed an assay for measuring c-erbB-2 amplification in DNA from human breast tumors. Protooncogene amplification has been consistently observed in human tumors and, although no direct correlation between gene amplification and the pathogenic mechanisms of tumorigenesis has been clearly established, in several cases detection of oncogene amplification can serve as a genetic marker for prognosis. In particular, several authors have reported a direct correlation between c-erbB-2 amplification in breast carcinoma and clinical outcome (15)(16)(17)(18).

In this study, we used the instrumentation and the fluorogenic probes of the Perkin-Elmer Cetus (Norwalk, CT) LS-50B TaqMan^TM System (19). The results were compared with those of a previously assessed competitive PCR method for measuring c-erbB-2 oncogene amplification (8)(9).

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
"taqman" principle and design of fluorogenic probes
The principle of the TaqMan LS-50B system is schematically summarized in Fig. 1 . Specific oligonucleotide probes are designed to anneal to the target between the two PCR primers (panel A, top). The probe contains 6-carboxy-fluorescein as the fluorescent reporter dye covalently linked to the 5' end. The quencher dye, 6-carboxy-tetramethyl-rhodamine, is usually covalently linked close to the 3' end. In addition, the probe contains a 3'-blocking phosphate group to prevent probe extension during PCR cycling (19). The closeness of the quencher to the reporter emitter means that the reporter fluorescence is suppressed, mainly by Förster-type energy transfer (20)(21) (Fig. 1B ). During PCR cycling, the probe specifically hybridizes to the corresponding template and then is cleaved via the 5'3' exonuclease activity of Taq DNA polymerase (19) (Fig. 1A ). This cleavage results in an increase of fluorescence emission of the 6-carboxy-fluorescein reporter dye, without affecting the emission of quencher dye (Fig. 1B ). This sequence of events occurs in each PCR cycle, in the enzymatic reaction, and in the PCR product accumulation. Because the exonuclease activity of the Taq polymerase acts only if the fluorogenic probe is annealed to the target (but cannot hydrolyze the probe free in solution), the increase of fluorescence is proportional to the amount of the specific PCR product.

View larger version (18K): [in this window] [in a new window]   Figure 1. Schematic representation of fluorescence emission by TaqMan probe reaction. During PCR annealing, the fluorogenic oligonucleotide specifically hybridizes to the corresponding template (A,top); the probe is then cleaved via the 5'3' endonucleolytic activity of Taq polymerase, during PCR extension (A, bottom). The release of the reporter dye (R) from the 5' end and its consequent separation from the quencher dye (Q) generates a specific fluorescence emission at 518 nm (B, bottom), but no signal is emitted when the probe is intact (B, top). The probe is then displaced from the target, and the polymerization of the strand continues.

The sequence of the probes utilized in this report to detect ß-actin, ß-globin, and c-erbB-2 genes is reported in Table 1 . Probes were designed according to the manufacturer's guidelines (19). Briefly, the TaqMan probe should (a) be 20–40 bases long, to ensure good hybridization and specificity of binding; (b) have a GC content of 40–60% and avoid long runs of a single nucleotide; and (c) neither hybridize nor overlap with the forward and reverse primers. It is important to design a probe that forms a more stable hybrid than do the PCR primers; thus, the melting temperature (T_m) of the probe should be at least 5 °C higher than that of the PCR primers. Synthesis and purification of the fluorogenic probes was performed by Perkin-Elmer Cetus. The detection system for ß-actin gene, provided by the manufacturer as "reference detection reagents" (code 401846), was used for preliminary assessment of the PCR conditions.

tumor samples and dna extraction
We used the proposed method to measure amplification of c-erbB-2 oncogene in 25 primary breast tumors (amplification range 1–22-fold), in which the presence of this genetic alteration had already been determined with a previously described competitive PCR procedure (8)(9), and in DNA specimens obtained from formalin-fixed, paraffin-embedded breast tumor. DNA was extracted from archived samples after deparafinization, as reported elsewhere (22), by a routine phenol–chloroform procedure (23).

Histopathological classification of the tumor samples was as follows: The tumor stage was pT1a in 2 cases, pT1b in 3, pT1c in 9, pT2 in 9, and pT4 in 2 cases; the nuclear grade was G3 in 14 cases, G2 in 9, and G1 in 2. Eighteen patients showed nodal involvement at the time of surgery.

pcr conditions
For PCR amplification, we utilized a Model 480 thermal cycler from Perkin-Elmer Cetus. The 10x PCR Buffer; the mononucleotides dATP, dCTP, dGTP, and dTTP; and the AmpliTaq DNA Polymerase were also from Perkin-Elmer. The PCR mix used for the TaqMan system was: 10 mmol/L Tris-HCl (pH 8.3), 50 mmol/L KCl, 1.5 mmol/L MgCl₂ (3.5 mmol/L MgCl₂ for the ß-actin gene), 0.2 mmol/L of each dNTP, 0.3 µmol/L of each primer, 0.2 µmol/L of each probe, and 1.25 U of AmpliTaq (Perkin-Elmer Cetus) in a 50-µL final volume. The PCR cycles for ß-globin and for c-erbB-2 were: 30 s at 94 °C, 30 s at 60 °C, and 30 s at 72 °C. For ß-actin amplification, the PCR conditions were: 30 s at 94 °C, 30 s at 54 °C, and 30 s at 72 °C. Primer sequences are reported in Table 1 .

fluorescence measurement and calculations
The fluorescence was measured with a luminescence spectrometer (Model LS-50B; Perkin-Elmer) equipped with a plate reader (see Fig. 2 ). We adopted a standard configuration for use in the three assays: 488 nm excitation wavelength; 518 and 580 nm emission wavelengths for the reporter and quencher dyes, respectively; and a 2-s integration time. After PCR cycling, a maximum of 40 µL from each sample or blank was transferred to a microwell.

View larger version (118K): [in this window] [in a new window]   Figure 2. (Top) The luminescence spectrometer (Perkin-Elmer Cetus LS-50B) used for fluorescence measurement of the TaqMan-generating system; (bottom) the 96-well plate reader.

As previously described [19], the fluorescent signal results from changes in the fluorescence emission intensity of the reporter dye after the cleavage of the probe. Interfering fluorescence fluctuations are normalized by applying two calculations. In the first, the quencher dye is a passive internal standard; we divide the emission intensity of the reporter dye by the emission intensity of the quencher dye for each reaction to give a ratio defined as the RQ+ value. Nonspecific effects, e.g., concentration changes resulting from volume fluctuations, are normalized by this ratio. Any other fluctuation, besides that attributable to PCR-related nuclease digestion, is normalized by taking the RQ+ value for a sample tube that contains all components (including target DNA) and subtracting from this the value of the "no-template" control tube, i.e., containing all of the same components except template and defined as RQ^-. This final RQ value (i.e., RQ+ - RQ^-) reliably indicates the magnitude of the signal generated by the given set of PCR conditions (19). Calculations and procedures for fluorescence signal acquisition are obtained with a personal computer connected on-line with the LS-50B luminescence spectrometer and equipped with a proprietary software specifically developed for the TaqMan technology.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
effect of pcr cycle number and dna quantity on taqman reaction: ß-actin calibration
The preliminary requirement of a PCR-based quantitative assay performed in the absence of an internal standard is to maintain the exponential phase of product accumulation, which is influenced by several known variables (number of cycles, DNA target concentration) and by certain unpredictable variables (mainly associated with the quality of the samples). In addition, in the method reported here, PCR product yield also depends on the efficiency of the cycling reaction of hybridization and on the cleavage of the fluorogenic probe. For these reasons, PCR conditions were previously assessed for the ß-actin gene (described as "reference detection reagents" in TaqMan principle and design of fluorogenic probes), with respect to the number of PCR cycles and the dose dependence of product accumulation. To verify the TaqMan PCR dependence on the number of PCR cycles, we amplified the ß-actin gene, starting with three quantities of normal target DNA (2.5, 10, and 40 ng) and using various numbers of PCR cycles. Evaluating the fluorescence emission (RQ+) associated with PCR product accumulation, we observed a general linear relationship between number of cycles and generated signal in the range 28–34 cycles for all three starter DNA quantities (Fig. 3 A). However, evaluation of the linear response of RQ measured at 28, 31, and 34 cycles, as related to the amount of target DNA, showed a better dose-dependent response in the range between 28 and 31 cycles (Fig. 3B ). Consequently, we performed the subsequent experiments with a standard 30-cycle PCR amplification.

View larger version (18K): [in this window] [in a new window]   Figure 3. Dependency of TaqMan fluorescence emission as a function of DNA target quantities and PCR cycle number: (A) ß-actin gene amplification, expressed as RQ+ values, performed with increasing numbers of PCR cycles, starting from three quantities of normal DNA and a no-template sample; (B) linear RQ response at 28, 31, and 34 PCR cycles for the same DNA template amounts.

taqman pcr assay conditions for ß-globin and c-erbb-2 genes
Using the fluorogenic probes for ß-globin and c-erbB-2 and the experimental conditions previously defined for the ß-actin gene, we obtained a linear relationship between the DNA concentration and the fluorescent signal (RQ) for ß-globin and c-erbB-2 genes in 2.5–20 ng of DNA target. Even if the linear response for the two genes showed different slope coefficients (mainly attributable to differences in efficiencies of fluorescence-generating reactions), the c-erbB-2/ß-globin fluorescence ratio was constant in this range of target concentrations of normal DNAs (Fig. 4 ).

View larger version (20K): [in this window] [in a new window]   Figure 4. Linear relationships between amounts of DNA target and fluorescence output, expressed as RQ, for ß-globin and c-erbB-2 genes. A 30-cycle amplification was performed on 2.5, 5, 10, and 20 ng of a normal DNA. The dotted line indicates the stability of c-erbB-2/ß-globin ratio at various DNA doses.

measuring c-erbb-2 amplification in breast tumor specimens
To determine the amplification extent of c-erbB-2 oncogene in breast tumor specimens, we tested 2.5 ng each of ß-globin and c-erbB-2 in different tubes but in the same run. Four replicates of human placental DNA were also assayed for both ß-globin and c-erbB-2 genes. For each unknown sample, we determined the RQ values for both genes as well as the c-erbB-2/ß-globin ratio. Finally, we divided this ratio by the mean c-erbB-2/ß-globin fluorescence ratio obtained in placental DNA samples. When c-erbB-2 amplification values were >10, the DNA sample being measured was diluted before the TaqMan PCR assay.

assay performance
To test the precision of TaqMan-based PCR assay, we measured in the same assay the RQ values for c-erbB-2 in 10 replicates of normal DNA, starting with two different template amounts (2.5 and 8 ng). The intraassay CV of the RQ values for c-erbB-2 in each template was 3.3% and 4.7%, respectively. The interassay precision in 6 different runs was 15.4% and 17.3%, respectively.

We tested the capacity of the TaqMan system to discriminate between different-fold c-erbB-2 amplifications by measuring the amplification in reconstituted samples obtained by serial dilutions in placental DNA of a DNA extracted from the SKBR.3 cell line carrying an 11-fold c-erbB-2 amplification (24). The results are reported in Fig. 5 .

View larger version (21K): [in this window] [in a new window]   Figure 5. Accuracy of the homogeneous PCR assay based on the TaqMan system. Decreasing amounts of DNA extracted from SKBR.3 cell line (T), containing ~11- fold-amplified c-erbB-2 gene, were mixed with increasing amounts of placental DNA (N) in the following proportions of T:N: A, 4:0; B, 3:1; C, 2:2; D, 1:3; E, 0:4. Reconstituted samples were designed to have a constant amount of DNA in each sample (2.5 ng) but different numbers of c-erbB-2 gene copies (from 11 to 1). We tested these reconstituted samples and plotted the expected values for c-erbB-2 amplification against the values derived from the reconstitution experiment. Linear regression analysis of the results is shown.

To verify the correspondence with classical hybridization techniques, we quantified c-erbB-2 amplification by the proposed method in four breast cancer cell lines (SKBR-3, ZR75.1, T47D, and MDA.MB.231) in which the oncogene amplification had been previously measured with Southern transfer analysis (Table 2 ).

Finally, we determined the amplification of c-erbB-2 in DNA extracted from 25 formalin-fixed, paraffin-embedded breast tumor samples in which the oncogene amplification had already been documented by a competitive PCR assay. The results obtained with the two PCR-based techniques showed a good correlation: r = 0.94, P <0.001 (Fig. 6 ).

View larger version (26K): [in this window] [in a new window]   Figure 6. Linear relation of c-erbB-2 amplification in 25 breast cancers as measured by the TaqMan procedure and by the competitive PCR.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In breast cancer, several different oncogenes (c-erbB-2, c-myc, and int-2) have been reported as potentially amplified (25). The prognostic value of these molecular lesions has been widely examined. In particular, in most reports, the amplification of c-erbB-2 has been considered prognostically unfavorable, being related to reduced survival and shorter relapse-free intervals in breast and ovarian carcinoma (15)(16)(26)(27)(28)—although conflicting results have been reported (18)(29)(30)(31). These differences can be explained by the high variability of semiquantitative assay procedures used in such studies, based mainly on Southern transfer or dot–blot analysis. New techniques, performed with standardized and well-controlled protocols, may represent an improved approach for understanding the clinical relevance of this and other quantitative molecular lesions in human cancers.

Accurate quantitative procedures for measuring nucleic acids, combined with the high sensitivity of PCR, are new and suitable tools for addressing specific requirements of diagnostic investigations (as in determinations of oncogene amplification) and are now becoming available for use in the clinical laboratory (32). However, up-to-date, independent, quantitative PCR methods usually require cumbersome postamplification procedures that can be additional sources of inaccuracy and contamination.

The novel procedure reported here allows accurate determination of PCR-amplified targets by a homogeneous technique. The specific signal derived by the use of a fluorogenic probe accumulates during PCR cycling and can easily be measured by end-point evaluation of specific fluorescence. The only difference between this system and other routine PCR procedures is the addition of a single reagent to the reaction mixture: a fluorogenic probe designed to anneal to the target sequence. In this sense, this system provides simultaneously (a) the desired information for quantitative evaluation and (b) qualitative recognition of the amplified target. The presence of a specific internal probe, carrying the signal-generating system, guarantees the specificity of the PCR product to be measured. Further, the intensity of the fluorescence signal allows the detection of PCR products in samples originally containing low quantities of DNA templates, even after a limited number of PCR cycles.

Oncogene amplification is a common quantitative alteration in tumoral DNA; therefore, we chose the increase of c-erbB-2 oncogene copy number as a suitable model for developing a quantitative TaqMan PCR assay. We first assessed the PCR conditions that would yield a linear response between DNA target doses and fluorescence signal from amplified products—the preliminary requirement for assessing a quantitative assay in the exponential phase of PCR. Using ß-actin, ß-globin, and c-erbB-2 fluorescent probes, we determined the range of both the number of PCR cycles and the amounts of DNA that should be used for maintaining the linear relationship between amplified target and PCR-generated product. These preliminary data were then applied to the development of the assay for c-erbB-2 amplification. Results for DNA extracted from paraffin blocks of 25 breast primary tumors, which provided only limited quantities of variable degraded DNA unsuitable for Southern transfer analysis, were compared with those obtained with an already developed competitive PCR procedure. This latter method is known to provide highly accurate and sensitive results, but the procedure (competitor construction, assessment of competitive PCR conditions, and measurement of PCR products) is quite complex. However, the TaqMan PCR assay described here represents a promising approach to obtaining reasonable accuracy for a quantitative PCR assay. The TaqMan PCR assay gave results comparable with those of Southern transfer analysis in DNA extracted from four breast cancer cell lines.

We conclude that the TaqMan procedure is easily applicable to qualitative applications in its standard configuration but, when the PCR conditions are well standardized and the exponential phase is well controlled, can also be successfully extended to quantitative applications. Our results showed a good agreement between the TaqMan assay and competitive PCR for determining the extent of c-erbB-2 amplification. Further, evaluation of precision and accuracy seems to indicate reliability of the TaqMan procedure for this measurement, apparently opening further improvements for quantitative PCR methods. However, further methodological studies will be necessary to confirm that the TaqMan methodology can be considered an accurate and sensitive quantitative PCR methodology.

	Acknowledgments

 
We are grateful to Perkin-Elmer Italia for providing fluorogenic probes and instrumentation used in this study. This work was supported by a grant of the University of Florence (Italy).

	Footnotes

 
Clinical Biochemistry Unit, Department of Clinical Physiopathology, University of Florence, Viale Pieraccini 6, 50134, Florence, Italy.

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Top Abstract Introduction Materials and Methods Results Discussion References

 

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