Absolute Quantification of the Alleles in Somatic Point Mutations by

Jul 28, 2011 - The detection and, particularly, the quantification of somatic mutations (acquired mutations) constitute a remarkable challenge in nucl...
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Absolute Quantification of the Alleles in Somatic Point Mutations by Bioluminometric Methods based on Competitive Polymerase Chain Reaction in the Presence of a Locked Nucleic Acid Blocker or an Allele-Specific Primer Alexandra Iliadi,† Margarita Petropoulou,† Penelope C. Ioannou,*,† Theodore K. Christopoulos,‡,§ Nikolaos I. Anagnostopoulos,^ Emmanuel Kanavakis,z and Jan Traeger-Synodinosz †

Laboratory of Analytical Chemistry, Department of Chemistry, Athens University, Athens 15771, Greece Department of Chemistry, University of Patras, Patras 26500, Greece § Foundation for Research and Technology Hellas, Institute of Chemical Engineering and High Temperature Chemical Processes (FORTH/ICE-HT), Patras 26504, Greece ^ Department of Clinical Haematology, General Hospital of Athens 'G. Gennimatas', Greece z Laboratory of Medical Genetics, Athens University, Athens 11527, Greece ‡

bS Supporting Information ABSTRACT: In somatic (acquired) point mutations, the challenge is to quantify minute amounts of the mutant allele in the presence of a large excess of the normal allele that differs only in a single base pair. We report two bioluminometric methods that enable absolute quantification of the alleles. The first method exploits the ability of a locked nucleic acid (LNA) oligonucleotide to bind to and inhibit effectively the polymerase chain reaction (PCR) amplification of the normal allele while the amplification of the mutant allele remains unaffected. The second method employs allele-specific PCR primers, thereby allowing the amplification of the corresponding allele only. DNA internal standards (competitors) are added to the PCR mixture to compensate for any sample-tosample variation in the amplification efficiency. The amplification products from the two alleles and the internal standards are quantified by a microtiter well-based bioluminometric hybridization assay using the photoprotein aequorin as a reporter. The methods allow absolute quantification of less than 300 copies of the mutant allele even in samples containing less than 1% of the mutant allele.

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he detection and, particularly, the quantification of somatic mutations (acquired mutations) constitute a remarkable challenge in nucleic acid analytical chemistry. First, contrary to the inherited mutations that are present in all the cells of an individual, an acquired mutation appears in certain cells of the body while the other cells contain only the normal allele. This leads to very low copy numbers of the mutant allele in a sample of genomic DNA (“minority” mutations). Second, in acquired mutations, the load of the mutant allele (allele burden) varies, greatly, depending on the number of affected somatic cells, and the challenge is to quantify the mutant allele in the presence of a large excess of the normal allele. On the contrary, in inherited mutations, the load of the mutant allele is predetermined (e.g., either 50% for a heterozygote or 100% for a homozygote for the mutation). Third, in somatic point mutations, the analytical methods must discriminate the mutant from the normal allele that differs only in a single nucleotide. Because the acquired mutations are considered as the primary cause of human cancer, the detection and quantification of the mutant allele is critical for diagnosis, prognosis, and monitoring of therapy.1,2 The rationale arises from the fact that the detection of the mutant allele signifies the presence of the affected cells and r 2011 American Chemical Society

its amount is related to the number of the affected cells in the body, i.e., to the severity of the disease. The first quantitative studies for the mutant allele were based on direct sequencing, but the detectability was low.3 Current methods are based on real-time polymerase chain reaction (PCR) with allele-specific primers or real-time PCR exploiting the 50 nuclease activity of Taq DNA polymerase.36 The advantage of the homogeneous fluorometric assays is that the detection is accomplished during amplification. However, costly equipment and expensive reagents (e.g., double labeled fluorescent oligonucleotides) are required. The BEAMing (beads, emulsion, amplification, and magnetics) technology was also applied to the digital quantification of the mutant allele burden. The technology is based on a combination of microemulsion PCR on magnetic beads followed by allele-specific oligonucleotide hybridization and laser-induced fluorescence detection on a flow cytometer.1 On the other hand, end-point bio(chemi)luminometric assays have found wide applications in DNA/RNA Received: March 31, 2011 Accepted: July 28, 2011 Published: July 28, 2011 6545

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Analytical Chemistry assays since they provide higher detectability, wider linear range, high throughput, and simpler instrumentation (no need for excitation light).710 Enzymes, along with chemiluminogenic substrates, or photoproteins have been employed as reporters in hybridization assays for the determination of PCR-amplified DNA,1113 determination of microRNA,14 and quantitative competitive PCR.15,16 Bio(chemi)luminometric assays for genotyping of point mutations (e.g., single-nucleotide polymorphisms) comprise three steps: (i) exponential amplification, usually by PCR, (ii) an allele-discrimination reaction, e.g., primer extension reaction17,18 or oligonucleotide ligation reaction,19 and (iii) detection of the reaction product by exploiting a bio(chemi)luminescent reporter. However, the aforementioned assays simply discriminate the three genotypes (normal, homozygote, and heterozygote) in inherited mutations without quantifying the amount of the mutant allele in the sample. Most recently, a bioluminometric assay for the relative quantification of the allele burden was reported with application to the oncogenic somatic point mutation JAK2 V617F20 Following PCR, two primer extension reactions were performed using allele-specific primers. The products were detected by a heterogeneous bioluminometric assay. The method enabled determination of the ratio of the mutant to normal alleles but not the absolute amount of the mutant allele. In the present work, we have developed two bioluminometric methods for the absolute quantification of the mutant allele in a somatic point mutation. The first method exploits the ability of a nonextendable locked nucleic acid (LNA) to inhibit effectively the PCR amplification of the normal allele while the amplification of the mutant allele remains unaffected. The second method employs allele-specific PCR primers, thereby allowing the amplification of the corresponding allele only. In both assays, absolute quantification of the mutant allele is achieved through coamplification of recombinant DNA internal standard(s) (DNA competitor(s)). The addition of the DNA internal standard compensates for any sample-to-sample variation in the yield of amplification. The amplified products from the target and internal standard are quantified by hybridization assays that are performed at microtiter wells and exploit the advantages of the photoprotein aequorin reporter. The ratio of the luminescence values for target DNA and DNA competitor is linearly related to the number of JAK2 V617F allele copies initially present in the sample. As a model, the two methods were applied to the absolute quantification of the JAK2 V617F mutant allele in human genomic DNA. In 2005, it was found that JAK2 V617F is strongly associated with the myeloproliferative disorders polycythemia vera and essential thrombocythemia.2124 In 2008, JAK2 V617F was included in the World Health Organization diagnostic markers for myeloproliferative disorders.25,26 Thus, reliable quantification of the JAK2 V617F allele is of major importance.

’ EXPERIMENTAL SECTION Information about the instrumentation, materials, clinical samples, PCR amplification, synthesis of mutant JAK2 DNA fragment, construction of recombinant DNA internal standards (DNA Competitors), and tailing of oligonucleotide probes with poly(dA) is included under Supporting Information. Absolute Quantification of the Mutant Allele by Competitive PCR in the Presence of a Locked Nucleic Acid Blocker (LNACoPCR). Competitive PCR. PCR was performed in a final

volume of 50 μL, containing 1 PCR buffer, 4 mM MgCl2, 200 μM dNTPs, 0.3 μM of each JAK2-F2 and JAK2-R(IC)-BIO primer, a

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constant amount (10 000 copies) of internal standard (ISM), 2.5 U AmpliTaq DNA polymerase (Stoffel fragment), 0.25 μM of LNA blocker, and 100 ng of genomic DNA. Cycling parameters were as follows: initial denaturation at 95 C for 3 min followed by 30 cycles of 95 C for 1 min, 55 C for 1 min, 72 C for 1 min, and a final extension step at 72 C for 8 min. The amplified DNA fragments were quantified by bioluminometric hybridization assay (see below). Bioluminometric Assay. Polystyrene microtiter wells were coated for 2 h at 42 C with 50 μL of 1.4 mg/L streptavidin diluted in phosphate-buffered saline, (NaCl 137 mM, KCl 2.7 mM, Na2HPO4 10 mM, KH2PO4 1.76 mM, pH = 7.4). Prior to use, the wells were washed three times with wash solution (50 mM TrisHCl, 150 mM NaCl, 0.2 mL/L Tween-20, 2 mM EDTA). A 2-μL aliquot of LNACoPCR product along with 48 μL of assay buffer (10 g/L BSA in sodium citrate 15 mM, NaCl 150 mM, and EDTA 2 mM, pH 7.5) were added into each well in duplicate. The binding of biotinylated PCR products to streptavidin was allowed to proceed for 15 min at room temperature under gentle shaking. The wells were washed three times with wash solution, and then, 50 μL of 0.1 M NaOH were added. After incubation for 5 min, the nonbiotinylated DNA strands were removed by washing the wells. A 50-μL aliquot of a solution containing 10 nM of normal or internal standard probes diluted in hybridization solution was added into the wells, and the hybridization reaction was allowed to proceed for 20 min at 42 C. The excess of probes was removed by washing the wells three times, and 50 μL of 22 nM aequorin-(dT)30 diluted in hybridization solution was added into the wells. Following 15 min incubation at room temperature with shaking, the excess of conjugate was removed by washing the wells as above. Finally, the activity of bound aequorin was measured by injecting 50 μL of triggering solution (25 mM CaCl2, 20 mM TrisHCl, pH 7.5) into each well and integrating the luminescence signal for 3 s. In the presence of LNA, the ratio, L/LIS, of the luminescence signals obtained from the mutant allele (L) and the mutant internal standard (LIS), after background subtraction, is used for the absolute quantification of the mutant allele via a calibration graph. In the absence of LNA, the ratio, L/LIS, of the luminescence signals obtained from both alleles (L) and the mutant internal standard (LIS), after background subtraction, is used for the absolute quantification of the total number of copies of both alleles via a calibration graph. The calibrators contain various copies of the mutant allele and normal allele in the presence of a fixed amount (10 000) of internal standard. The calibration curve is the plot of L/LIS versus the number of copies. Absolute Quantification of the Total Copy Number of the Alleles by Competitive PCR (CoPCR). The procedure is exactly as above but in the absence of LNA blocker. Absolute Quantification of the Alleles by Allele-Specific Competitive PCR (ASCoPCR). For quantification of the mutant allele, the amplification reaction was performed with primers JAK2-F2 (common for both alleles) and J2-AS1M-BIO (specific for the mutant allele). The final volume was 50 μL containing 1U Hotstar Plus DNA polymerase, 1 PCR buffer, 2 mM MgCl2, 200 μM dNTPs, 0.3 μM of each primer, ∼100 ng of genomic DNA, and 5000 copies of ISM plasmid. The thermal cycling protocol included the following steps: 95 C/5 min followed by 27 cycles of 95 C/30 s, 55 C/30 s, 72 C/30 s, and a final extension step at 72 C for 8 min. Quantification of the normal allele was performed with an allele-specific PCR as above, but the reaction mixture contained 6546

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the J2-AS1N-BIO primer instead of J2-AS1M-BIO and the ISN plasmid instead of the ISM. The amplified DNA fragments were quantified by a bioluminometric hybridization assay as above except that 1 μL of ASCoPCR product was used instead of 2 μL. The ratios, L/LIS, of the luminescence signals obtained from the mutant allele or normal allele (L) and the corresponding internal standards (LIS), after background subtraction, is used for the absolute quantification of the mutant allele or normal allele via a calibration graph. The calibrators contain various copies of the mutant allele and normal allele in the presence of a fixed amount (5000) of each internal standard. The calibration curve is the plot of L/LIS versus the number of copies of the mutant allele or normal allele, respectively.

’ RESULTS AND DISCUSSION Absolute Quantification of the Mutant Allele via Competitive PCR in the Presence of a LNA Blocker (LNACoPCR).

We designed an LNA oligonucleotide homologous to the region of JAK2 normal allele that flanks the mutation. LNA blocker is a 15-mer comprising 9 LNA bases and 3 mismatched bases, at the 30 end, to prevent extension by DNA polymerase. A mutated form of Taq DNA polymerase, termed Stoffel fragment27 was used, which lacks both 50 to 30 and 30 to 50 exonuclease activity. LNA blocker inhibits amplification of the normal allele without affecting the amplification of the mutant allele.28,29 LNA oligonucleotides possess higher thermal stability and selectivity over their natural counterparts.30 This property is due to a structural feature of these analogs, an additional methylene bridge linking the 20 hydroxyl group to the 40 carbon of the ribose. The bridge locks the sugar ring in a C30 -endo conformation typical of RNA conformation.31,32 A schematic presentation of the LNA-mediated quantitative competitive polymerase chain reaction (LNACoPCR) is shown in Figure 1. Each sample is subjected to two PCR, in the presence and absence of the LNA blocker. Both PCR mixtures contain a constant amount of recombinant mutant DNA internal standard (ISM) whose amplification is not affected by the LNA blocker. Normal and mutant alleles are amplified by the same pair of primers producing 447 bp fragments. The amount of amplification product generated in the absence of LNA blocker corresponds to the total number of copies of both alleles (normal and mutant) in the sample. The amount of amplification product obtained in the presence of LNA blocker corresponds to the amount of mutant allele in the sample. We performed a series of experiments to determine the minimum concentration of the LNA blocker that prevents amplification of the normal allele but has no effect on the amplification of the mutant allele. The effect of LNA blocker concentration was studied in the range of 3.9 to 1000 nM. The PCR products were subjected to agarose gel electrophoresis and ethidium bromide staining. Prior to the bioluminometric assay, the products were diluted 25-fold. It was found that the blocking effect on the amplification of the normal allele increases with increasing LNA probe concentration and, practically, no amplification occurs at LNA blocker concentration of 250 nM (especially after the 25-fold dilution of the product prior to the bioluminometric assay). At the same time, no effect on the amplification of mutant allele is observed. At a higher LNA blocker concentration (1000 nM), the amplification of the mutant allele is also inhibited (Figure 1).

Figure 1. (A) Outline of the principle of LNA-mediated quantitative competitive PCR (LNACoPCR). Each sample is subjected to two PCR reactions, one in the presence and, the second, in the absence of LNA oligonucleotide. A nonextendable LNA oligonucleotide, when present, binds tightly to a region of the normal allele that corresponds to the point mutation. The LNA sequence blocks the amplification of the normal allele without affecting the amplification of the mutant allele. Both reactions use the same set of primers. (B) Electropherogram showing the effect of the concentration of the LNA blocker on the amplification of the mutant and the normal allele. The faint band observed in all lanes is due to the primer dimers. (C) Schematic illustration of the principle of the hybridization assay. Biotinylated PCR product is captured on streptavidin-coated microtiter wells. Oligonucleotide probes comprising a target-specific sequence and a poly(dA) tail, hybridize both with the biotinylated PCR product and the oligo(dT)-conjugated aequorin. The bioluminescence of bound aequorin is measured by the addition of Ca2+. B = biotin; SA = streptavidin; Aeq = aequorin.

The principle of the microtiter well-based hybridization assay is illustrated in Figure 1. The downstream primer is biotinylated at the 50 end. The biotinylated 447 bp amplification products (target and internal standard) are captured in the same well by immobilized streptavidin. After removing the nonbiotinylated strand with NaOH, the immobilized single-stranded DNA is hybridized with probes that are specific either for the target or for the IS. The specific probes consist of a unique 23-bp segment complementary to the analyte and a poly(dA) tail. The hybrids are detected bioluminometrically by reacting with (dT)30-conjugated photoprotein aequorin. The luminescence of bound aequorin is measured within 3 s by simply adding Ca2+. The hybridization assay is complete in ∼1 h. To verify the presence of the characteristic 23-bp sequences in ISN and ISM and to confirm that the amplification products from the target allele and the IS were distinguishable by hybridization, we performed PCR on two samples containing 30 000 copies of either target or IS plasmid. Each PCR product (500 pM) was then analyzed in triplicate by hybridization with both probes, i.e., the target-specific and the IS-specific probe. The luminescence signals (L) obtained from amplified target plasmid assayed with the target and IS specific probes were 416.6 and 1.9, respectively. The amplified IS plasmid tested with the target and IS probes gave signals of 2.4 and 334.9, respectively. These data prove the specificity of the target and IS probes for their cognate targets. The low luminescence signals obtained when PCR products were tested with the noncomplementary probes reflect the nonspecific binding of the probe and the (dT)30-aequorin to the solid phase (typical readings for the assay blank). This experiment confirmed that cross-hybridization between targets and probes did not occur and that the IS solution was free of 6547

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Figure 2. (A) Calibration graphs for the bioluminometric hybridization assays of the 447-bp amplification products from the mutant allele (circles) and the internal standard (squares) generated by LNACoPCR. (B) Calibration graphs for the bioluminometric hybridization assays of the 240-bp amplification products from the mutant allele (M, circles) and the internal standard (IS, squares) generated by ASCoPCR. Stock solutions of each amplified DNA were prepared by PCR, and the DNA concentration was determined by scanning densitometry of ethidium bromide-stained agarose gels. Various dilutions of the stock solutions were then analyzed by the microtiter well-based hybridization assays as described in the Experimental Section.

contamination from the target DNA and/or its amplification product (and vice versa). The performance of the hybridization assay was studied using PCR-amplified products from the target (normal allele) and the IS whose concentrations were determined by densitometry of images of ethidium bromide-stained gels using the ΦX174 DNA marker as calibrator. The detectability and linear range of the hybridization assays were established by analyzing serial dilutions of stock solutions containing either amplified target (normal allele) or amplified IS. The signal-to-background (S/B) ratios at the 4 pM level (200 amol/well) of amplified target or IS are 2.3 and 3.5, respectively. The linear range of the assays extends up to 1000 pM (Figure 2). The reproducibility of the bioluminometric hybridization assay was assessed by analyzing samples containing 4, 37, and 334 pM of amplified products. The coefficients of variation (CVs) were 11.8, 9.9, and 7.6%, respectively (n = 5). For the absolute quantification of JAK2 mutant allele by the LNA-mediated competitive PCR, a series of calibrators containing 50 to 50 000 copies of the mutant allele along with a fixed amount (10 000 copies) of ISM were prepared by serial dilutions of plasmid DNA in a diluent containing 2000 copies/μL of ISM. A 5-μL aliquot of each calibrator was subjected to LNA CoPCR. Amplified products (2 μL) were analyzed in duplicate by the bioluminometric hybridization assays using target-specific and IS-specific probes. The calibration graph, i.e., the plot of the ratio of the luminescence signals (background subtracted), L/LIS, obtained from the target and internal standard, respectively, versus the number of the copies of the mutant allele, is presented in Figure 3. The background is defined as the signal obtained in the absence of target DNA in the PCR mixture. The overall reproducibility of the method (including PCR and hybridization assay) was assessed by analyzing samples containing 50, 500, 5000, and 50 000 copies of the mutant allele in the presence of 10 000 copies of IS (n = 3). The %CVs obtained for the L/LIS signal ratios were 25, 14.3, 6.9, and 9.9, respectively. The corresponding %CVs obtained for the estimated copy numbers of the allele were 28.9, 18.1, 7.5 and 9.6, respectively. The S/B signal ratio at the level of 500 copies of the mutant allele was found to be 4.0. The accuracy of the proposed method was evaluated by determining the number of copies of the mutant allele in synthetic

Figure 3. Calibration graphs for the absolute quantification of the mutant allele by LNACoPCR (squares corresponding to the left Yaxis) and ASCoPCR (circles corresponding to the right Y-axis). In each case, the ratio, L/LIS, of the luminescence values for the mutant allele and the internal standard is plotted against the absolute number of copies of the mutant allele in the sample prior to amplification. For LNACoPCR, the 30 cycles were performed and 2-μL aliquots of the PCR product were analyzed in duplicate by the hybridization assay. For ASCoPCR, 27 cycles were performed and 1-μL aliquots of the PCR product were analyzed in duplicate by the hybridization assay.

mixtures containing a total of 30 000 copies of both alleles (normal plus mutant) with a varying number of copies of the mutant allele. A fixed amount of 10 000 copies of ISM was added into each mixture prior to PCR. Aliquots (2 μL) of the PCR products were analyzed in triplicate by the hybridization assay. The total number of copies of both alleles and the number of copies of the mutant allele in each sample were determined using the L/LIS and LLNA/LIS ratios, respectively, and the calibration curve. LLNA, L, and LIS are the luminescence values obtained from each PCR product (with and without LNA), and the internal standard, respectively. The results are presented in Figure 4. It is observed that the number of the copies of the mutant allele determined by the proposed high-throughput LNACoPCR method is in close agreement with the nominal 6548

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Figure 4. Evaluation of the LNACoPCR and ASCoPCR methods by quantification of synthetic mixtures containing a total number of 30 000 copies of normal and mutant plasmids with a varying copy number of mutant plasmid. Aliquots of the PCR products were analyzed in duplicate by the hybridization assays. The results obtained from LNACoPCR and ASCoPCR (“copies found”) are plotted versus the nominal values of the synthetic mixtures (squares). The triangles show the experimentally determined total number of copies (mutant plus normal alleles) for each synthetic mixture. The dash line indicates the level of 30 000 copies.

values of the synthetic mixtures. Also, in Figure 4 can be seen that the total number of copies of both alleles as determined by the LNACoPCR is very close to the nominal value of 30 000 throughout the entire range of mutant allele copies. It is worth noting that the 300 copies of mutant allele in the mixtures correspond to an allele burden of 1% only, which shows the high detectability of the proposed method. Absolute Quantification of the Alleles via Allele-Specific Competitive PCR (ASCoPCR). The proposed ASCoPCR method comprises an allele-specific amplification reaction for each allele producing a 240 bp fragment of the JAK2 gene. The primer pair for each reaction consists of a common upstream primer and an allele-specific downstream primer. The normal (N) and mutant (M) primers carry at their 30 -end a nucleotide complementary to the normal and mutant allele, respectively. Both allele-specific primers are biotinylated at their 50 end. The reactions are carried out in the presence of a fixed amount (5000 copies) of either ISN or ISM. Competitive DNA internal standards for normal and mutant allele differ only in a 23-bp segment from their cognate target sequences, thereby enabling recognition by hybridization. The 240-bp products of ASCoPCR were analyzed by the bioluminometric hybridization assay as described above for the LNACoPCR products (see Figure 1). The detectability, linear range, and reproducibility of the hybridization assay for the 240 bp amplification fragments were established by analyzing serial dilutions of the stock solutions of amplified target and IS. A S/B ratio of 2 was obtained at 1.4 pM of amplified target and IS (67 amol/well). The analytical range of the assays extends up to 1000 pM (Figure 2). The reproducibility of the hybridization assay was assessed by analyzing samples containing low (1.4 pM), medium (37 pM), and high (1000 pM) concentrations of amplified fragments. The CVs were 12.2, 7.3, and 3.3% for both normal and mutant allele sequences, respectively (n = 3). Taking advantage of the high sensitivity of the proposed bioluminometric assay for the quantification of PCR products as well as the higher amplification efficiency of the HotStar Taq Plus DNA polymerase compared with Amplitaq DNA polymerase Stoffel fragment, we decreased the number of amplification cycles without compromising the detectability of the method. For this purpose, mixtures containing 30 to 30 000 copies of the

normal allele, along with a fixed amount (5000 copies) of ISN were subjected to PCR with number of cycles varying from 25 to 30. As low as 30 copies of the mutant allele were detectable after 30 cycles of amplification, and 117 copies were detectable after 27 cycles. For ASCoPCR, 27 cycles provide satisfactory detectability. Indeed, 117 copies correspond to ∼0.4% of mutant allele assuming that 100 ng of genomic DNA contains 28 600 human genome copies. For the absolute quantification of the alleles by ASCoPCR, calibrators containing 30 to 30 000 copies of either the mutant or the normal allele along with a constant amount (5000 copies) of ISM or ISN were used for the construction of calibration curves. Calibrators were prepared daily by serial dilutions of stock solutions of target DNA (mutant or normal) and ISM or ISN in 1 PCR buffer. PCR products (1 μL) were analyzed by the hybridization assay using specific probes. A calibration graph presented as a plot of the ratio of the luminescence signals L/LIS obtained from the target and the internal standard versus the copy number of the mutant allele prior to PCR is shown in Figure 3. The overall reproducibility of the ASCoPCR, including PCR and hybridization assay, was assessed by analyzing solutions containing 117, 469, 1875, 7500, and 30 000 copies of the mutant allele (n = 3). The %CVs obtained were 15, 8.0, 9.0, 4.0, and 14%, respectively. As low as 117 copies of the mutant allele in a total amount of 30 000 copies of both alleles (0.4%) could be detected with a signal-to-background ratio (S/B) of 3.7. The accuracy of the proposed method was also evaluated by quantifying the mutant allele copy number in synthetic mixtures containing a constant amount (30 000 copies) of normal and mutant plasmids at various ratios. A constant amount (5000 copies) of ISN or ISM was added into each PCR mixture. Aliquots (1 μL) of the PCR products were analyzed in duplicate by the hybridization assays. The number of the copies of normal and mutant alleles in each sample was estimated using the corresponding L/LIS ratios and the calibration curve for each target. The LM/LISM ratio and the LN/LISN ratio give the number of copies of the mutant and normal allele, respectively. LM, LN, LISM, and LISN are the luminescence signals obtained from the mutant allele, normal allele, ISM, and ISN, respectively. The results are presented in Figure 4. The number of copies represents the mean of three values. It is observed that the number of 6549

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Figure 5. Analysis of 37 clinical samples and comparison of the two new methods of absolute quantification (LNACoPCR and ASCoPCR) with a recently published method20 of relative quantification of the mutant allele (PEXT). Extraction of genomic DNA, PCR, and detection of products by hybridization assay were all performed as described in the Experimental Section. The allele burden was calculated as the ratio of the copies of the mutant allele to the total copies of both alleles in the sample (expressed as a percentage). The results of the proposed methods are compared with those obtained by PEXT reaction assay.

copies of the mutant allele is in concordance with the number of the copies added. Also, the estimated total number of copies of both alleles is close to the nominal value of 30 000. Genotyping of Clinical Specimens. The proposed LNA CoPCR and ASCoPCR assays were evaluated by the determination of the mutant allele burden in 37 blood samples. Extraction of genomic DNA, PCR, and detection of the products by hybridization assay were all performed as described in the Experimental Section. The mutant allele burden was calculated as the ratio of the copies of the mutant allele to the total copies of the two alleles (expressed as a percentage). In Figure 5, the results obtained by the two new methods are compared directly with a recently reported method that provides only a relative quantification and is based on a primer extension (PEXT) reaction.20 The results of the proposed methods are in good agreement with those obtained by the PEXT method.

’ CONCLUSIONS The LNA oligonucleotide inhibits effectively the amplification of the normal allele and allows the quantification of very low copy number of mutant allele in the presence of a large excess of normal allele. The primers for LNACoPCR do not have to be allele specific. It was also shown that the use of specific primers for ASCoPCR provides effective discrimination of the two alleles. The addition of internal standards in the PCR mixtures compensates for any sample-to-sample variations in the efficiency of the amplification reaction, thereby ensuring the accuracy and robustness of the proposed methods. The PCR products are quantified by rapid and highly sensitive bioluminometric hybridization assays that are performed in microtiter wells, thus facilitating automation for use in the routine laboratory. The hybridization assay is complete within ∼1 h after PCR. The methods allow absolute quantification even in samples containing less than 1% of the mutant allele. To conclude, the novelty and the advantages of the proposed method are as follows: (a) the concept of competitive PCR is exploited for the absolute quantification of the mutant and normal alleles in single-point mutations using properly designed recombinant DNA internal standards; (b) the combination of the above principle with a high-throughput and highly sensitive bioluminometric assay; (c) the considerably lower cost of

instrumentation and reagents compared to other methods, such as real-time PCR and BEAMing technology; (d) the incorporation of the LNA probes, in the aforementioned competitive PCR, simplifies the method because it does not require two allelespecific primers.

’ ASSOCIATED CONTENT

bS

Supporting Information. Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*Phone: +30 210-7274574. Fax: +30 210-7274750. E-mail: [email protected].

’ ACKNOWLEDGMENT A.I. and M.P. contributed equally to this work. ’ REFERENCES (1) Diehl, F.; Schmidt, K.; Durkee, K. H.; Moore, K. J.; Goodman, S. N.; Shuber, A. P.; Kinzler, K. W.; Vogelstein, B. Gastroenterology 2008, 135, 489–498. (2) Wadleigh, M.; Tefferi, A. Int. J. Hematol. 2010, 91, 174–179. (3) Rapado, I.; Albizua, E.; Ayala, R.; Hernandez, J.-A.; GarciaAlonso, L.; Grande, S.; Gallardo, M.; Gilsanz, F.; Martinez-Lopez, J. Ann. Hematol. 2008, 87, 741–749. (4) Poodt, J.; Fijnheer, R.; Walsh, I. B. B.; Hermans, M. H. A. Hematol. Oncol. 2006, 24, 227–233. (5) Lippert, E.; Girodon, F.; Hammond, E.; Jelinek, J.; Reading, N.-S.; Fehse, B.; Hanlon, K.; Hermans, M.; Richard, C.; Swierczek, S.; Ugo, V.; Carillo, S.; Harrivel, V.; Marzac, C.; Pietra, D.; Sobas, M.; Mounier, M.; Migeon, M.; Ellard, S.; Kroger, N.; Herrmann, R.; Prehal, J. T.; Skoda, R. C.; Hermouet, S. Haematologica 2009, 94, 38–45. (6) Lucia, E.; Martino, B.; Mammi, C.; Vigna, E.; Mazzone, C.; Gentile, M.; Qualtieri, G.; Bisconte, M.-G.; Naccarato, M.; Gentile, C.; Lagana, C.; Romeo, F.; Neri, A.; Nobile, F.; Morabito, F. Leuk. Lymphoma 2008, 49, 1907–1915. (7) Christopoulos, T. K.; Ioannou, P. C.; Verhaegen, M. Photoproteins in Nucleic Acids Analysis. In Photoproteins in Bioanalysis; 6550

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dx.doi.org/10.1021/ac200810h |Anal. Chem. 2011, 83, 6545–6551