Coamplification at lower denaturation temperature pcr increase mutation detection selectivity of taq

Coamplification at Lower Denaturation Temperature–PCR Increases Mutation-Detection Selectivity of TaqMan-BasedReal-Time PCRJin Li,1Lilin Wang,1Pasi A.Ja¨nne,2and G.Mike Makrigiorgos1*BACKGROUND:DNA genotyping with mutation-specific TaqMan®probes(Applied Biosystems)is broadly used in detection of single-nucleotide poly-morphisms but is less so for somatic mutations because of its limited selectivity for low-level mutations.We re-cently described coamplification at lower denaturation temperature–PCR(COLD-PCR),a method that ampli-fies minority alleles selectively from mixtures of wild-type and mutation-containing sequences during the PCR.We demonstrate that combining COLD-PCR with TaqMan technology provides TaqMan genotyping with the selec-tivity needed to detect low-level somatic mutations. METHODS:Minor-groove binder–based or common TaqMan probes were designed to contain a nucleotide that matches the desired mutation approximately in the middle of the probe.The critical denaturation tem-perature(T c)of each amplicon was then experimen-tally determined.COLD-PCR/TaqMan genotyping was performed in2steps:denaturation at the T c,fol-lowed by annealing and extension at a single tempera-ture(fast COLD-PCR).The threshold cycle was used to identify mutations on the basis of serial dilutions of mutant DNA into wild-type DNA and to identify TP53 (tumor protein p53)and EGFR[epidermal growth fac-tor receptor(erythroblastic leukemia viral(v-erb-b) oncogene homolog,avian)]mutations in tumors. RESULTS:COLD-PCR/TaqMan genotyping identified GϾA mutations within TP53exon8(codon273mu-tation hot spot)and CϾT mutations within the EGFR gene(drug-resistance mutation T790M)with a selec-tivity improvement of15-to30-fold over regular PCR/ TaqMan genotyping.A second round of COLD-PCR/ TaqMan genotyping improved the selectivity by another15-to30-fold and enabled detection of1mu-tant in2000wild-type e of COLD-PCR/Taq-Man genotyping allowed quantitative identification of low-level TP53and T790mutations in colon tumor samples and in non–small-cell lung cancer cell lines treated with kinase inhibitors. CONCLUSIONS:The major improvement in selectivity provided by COLD-PCR enables the popular TaqMan genotyping method to become a powerful tool for de-tecting low-level mutations in clinical samples.©2008American Association for Clinical ChemistryMutation detection plays a key role in the diagnosis, treatment,and prognosis assessment of cancer patients (1).Methods used for mutation detection include se-quencing(2,3),RFLP analysis(4),MALDI-TOF anal-ysis(5),denaturing HPLC/Surveyor™(6,7),ligation-mediated PCR(8),high-resolution melting(9,10), peptide nucleic acid(PNA)3-locked nucleic acids(11), antiprimer quenching real-time PCR(12,13),Scor-pion primers(14),molecular beacons,and methods based on TaqMan®probes(Applied Biosystems) (15,16).Because of its simplicity and speed,TaqMan genotyping is frequently used as an end-point ap-proach(17).The reaction consists of2primers and2 probes that match to either the wild-type or mutant allele.The polymorphic nucleotide is usually designed to be in the middle third of the probe,which is labeled with a reporter molecule at the5Јend and with a non-fluorescent quencher at the3Јend.Modifications of the TaqMan probe with minor-groove binders (MGBs)(18)or locked nucleic acids(19)increase the probe’s T m(temperature at which50%of the probe is denatured from the template)to allow the design of shorter probes and better discrimination between mu-tant and wild-type alleles.The selectivity limit of Taq-Man genotyping is the detection of mutant alleles present at an abundance of approximately10%–20%1Department of Radiation Oncology,Divisions of Genomic Stability and DNA Repair,and Medical Physics,and2Department of Medical Oncology,Lowe Center for Thoracic Oncology,Dana Farber Cancer Institute,Harvard Medical School,Boston,MA.*Address correspondence to this author at:Brigham and Women’s Hospital, Level L2,Radiation Therapy,75Francis St.,Boston,MA02115,USA.Fax(617) 587-6037;e-mail mmakrigiorgos@.Received June24,2008;accepted October14,2008.Previously published online at DOI:10.1373/clinchem.2008.1133813Nonstandard abbreviations:PNA,peptide nucleic acid;MGB,minor-groove binder;T m,temperature at which50%of the probe is denatured from the complementary strand;COLD-PCR,coamplification at lower denaturation tem-perature–PCR;T c,critical denaturation temperature;NSCLC,non–small-cell lung cancer;Taq MAMA,allele-specific PCR-based TaqMan genotyping.Clinical Chemistry55:4748–756(2009)Molecular Diagnostics and Genetics 748of that of the wild-type allele(17,20).Because the fre-quencies of somatic mutations can often be lower (6,21),this limit poses problems for the use of Taq-Man genotyping in screening for somatic mutations in tumor surgical samples or bodily fluids that are often contaminated with wild-type alleles.We recently described a new form of PCR,coam-plification at lower denaturation temperature–PCR (COLD-PCR),which preferentially enriches“minority alleles”from mixtures of wild-type and mutation-containing sequences,irrespective of where a mutation lies in the sequence(22).COLD-PCR is based on the observations that(a)for each DNA sequence there is a critical denaturation temperature(T c)that is lower than the T m of the target sequence and below which PCR efficiency drops abruptly,and(b)T c is dependent on the DNA sequence.DNA amplicons differing by a single nucleotide have substantially different and re-producible amplification efficiencies when the PCR de-naturation temperature is set to the T c.These features are exploited during PCR amplification to selectively enrich minority alleles that differ by one or more nu-cleotides at any position in a given sequence.Conse-quently,COLD-PCR amplification of genomic DNA yields PCR products that contain high percentages of variant alleles,thus permitting their detection.We have demonstrated that COLD-PCR improves the se-lectivity of RFLP analysis,denaturing HPLC/Surveyor, Sanger sequencing,pyrosequencing,and MALDI-TOF–based mutation detection by one to two orders of magnitude(22).We demonstrate that combining COLD-PCR with the TaqMan genotyping method provides a major improvement in the latter’s ability to quantitatively detect low-level somatic mutations in tumor samples in a real-time format.Materials and MethodsSOURCE OF GENOMIC DNAReference human male genomic DNA was purchased from Promega and used as wild-type DNA in dilution experiments with mutation-containing DNA.Genomic DNA from SW480and4lung adenocarcinoma cell lines(H1975,H820,PC9GR,and H3255GR)were pur-chased from the ATCC.The H3255GR cell line was developed by exposing H3255cells to serially increas-ing concentrations of gefitinib for6months until the cells were able to proliferate in100nmol/L gefitinib with growth kinetics similar to those of untreated cells (23).Similarly,the PC9GR cell line was derived by ge-fitinib treatment of PC9cells(24).Snap-frozen colon tumor samples were obtained from the Massachusetts General Hospital Tumor Bank following Internal Re-view Board approval.DNA was extracted from cell lines and tumor samples with the DNeasy Blood&Tis-sue Kit(Qiagen).Primers were synthesized by Inte-grated DNA Technologies.SINGLE-ROUND COLD-PCR/TaqMan GENOTYPINGCOLD-PCR/TaqMan real-time genotyping for the T790M mutation encoded by EGFR exon20.See (25,26)for further details.Real-time PCR reactions were performed directly with70ng genomic DNA in the presence of0.2␮mol/L regular TaqMan probe(5Ј–6-FAM-CAT GAG CTG CAT GAT GAG CTG-BHQ-1–3Ј)or0.1␮mol/L MGB TaqMan probe(5Ј–6-FAM-TGA GCT GCA TGA TGA GC-MGBNFQ–3Ј)that fully matches the mutation-containing sequence on DNA from H1975cells that encodes the T790M muta-tion(mutation is underlined).The final concentra-tions of the other reagents were as follows:1ϫGoTaq Flexi Buffer(Promega),1ϫGoTaq Flexi DNA Poly-merase(Promega),0.2mmol/L of each deoxynucleo-side triphosphate,0.2␮mol/L forward primer(5Ј–TGATGGCCAGCGTGGAC–3Ј),0.2␮mol/L reverse primer(5Ј–CAGGAGGCAGCCGAAGG–3Ј),and2.5 mmol/L MgCl2.The size of the PCR amplicon is104 bp.Fast COLD-PCR cycling was performed on a Ce-pheid SmartCycler™machine as follows:95°C for 120s;20cycles of95°C for15s and60°C(fluorescence reading on)for30s;and30cycles of88°C for15s and 60°C(fluorescence reading on)for30s.The88°C T c for this amplicon was determined experimentally,as described previously(22).In brief,a set of PCR reac-tions were performed at gradually decreasing denatur-ation temperatures(0.3°C steps starting from the T m), and the lowest denaturation temperature that repro-ducibly yielded a PCR product was chosen. Quantification of T790M mutations in lung adenocarci-noma cell lines with COLD-PCR/TaqMan genotyping. We first used regular PCR with an intercalating dye on a104-bp EGFR4[epidermal growth factor receptor (erythroblastic leukemia viral(v-erb-b)oncogene ho-molog,avian)]amplicon to quantify the copy number of EGFR exon20;the PCR was carried out independent of the presence or absence of a mutation.We used0.1ϫLCGreen dye(Idaho Technology)in this reaction without a TaqMan probe.The PCR cycling conditions were95°C for120s,and40cycles of95°C for15s and60°C(fluo-rescence reading on)for60s.We also used serial dilutions of known concentrations of reference DNA as a calibra-tion reference to quantify the copy numbers of DNA from non–small-cell lung cancer(NSCLC)cell lines.4Human genes:EGFR,epidermal growth factor receptor(erythroblastic leukemia viral(v-erb-b)oncogene homolog,avian);TP53,tumor protein p53.Real-Time COLD-PCRClinical Chemistry55:4(2009)749After determining EGFR allele copy numbers,we tested cell line DNA containing equivalent numbers of EGFR exon 20copies with COLD-PCR/TaqMan geno-typing to quantify the relative amounts of T790M mu-tations.Our assessment of the amount of mutant T790M allele as a percentage of the wild-type allele with COLD-PCR/TaqMan genotyping was based on a cali-bration curve of serial dilutions of known amounts of mutation-containing DNA added to wild-type DNA.COLD-PCR/TaqMan real-time genotyping of the G ϾA mutation in codon 273of the TP53exon 8fragment .See (27)for further details.Real-time PCR reactions were performed directly from 20ng genomic DNA in the presence of 0.2␮mol/L of a TaqMan probe (5Ј–6-FAM-TTT GAG GTG CAT GTT TGT GCC-BHQ-1–3Ј)that fully matches the mutation-containing se-quence in DNA from SW480cells (mutation is underlined).The final concentrations of the other re-agents were as follows:1ϫGoTaq Flexi Buffer,1ϫGoTaq Flexi DNA Polymerase,0.2mmol/L of each de-oxynucleoside triphosphate,0.2␮mol/L forward primer (5Ј–TGG TAA TCT ACT GGG ACG–3Ј),0.2␮mol/L reverse primer (5Ј–CGG AGA TTC TCT TCC TCT–3Ј),and 3mmol/L MgCl 2.The size of the COLD-PCR amplicon was 87bp,with a T c of 83.5°C defined experimentally as described above.The fast COLD-PCR cycling conditions were as follows:95°C for 120s;25cycles of 95°C for 15s and 58°C (fluores-cence reading on)for 60s;and 25cycles of 83.5°C for 15s and 58°C (fluorescence reading on)for 60s.Experiments were repeated at least 5times in inde-pendent experiments.PRINCIPLE OF COLD-PCR/TaqMan GENOTYPINGCOLD-PCR can be carried out in 2formats,full COLD-PCR and fast COLD-PCR,depending on whether it is necessary to detect all mutations compre-hensively or to detect specific T m -reducing mutations in a rapid and highly selective fashion (22).The com-bination of full COLD-PCR with TaqMan genotyping can be applied for T m -increasing mutations such as A:T ϾG:C or T:A ϾG:C or for T m -decreasing muta-tions;however,the T m of a DNA sequence is reduced for the great majority of mutations encountered in can-cer samples (28),including the T790M mutation (i.e.,C ϾT,EGFR exon 20)and the codon 273mutation (G ϾA,TP53exon 8)examined in this investigation.In view of the simplicity,speed,and high mutation en-richment achieved via fast COLD-PCR,we focused on developing the combination of fast COLD-PCR with TaqMan genotyping to detect T m -reducing mutations.Because the present application is aimed at detect-ing low-level mutant alleles,the COLD-PCR/TaqMan reaction uses a single TaqMan probe specific for the mutant allele,in which the mutation is placed approx-imately in the middle of the probe (i.e.,there is no need for a second TaqMan probe to detect the wild-type al-lele,as in conventional TaqMan genotyping).The cy-cling program includes approximately 20–25regular PCR cycles to build-up the PCR product,followed by a switch to a 2-step PCR consisting of denaturing at T c and then lowering to a single temperature for both primer annealing and extension (Fig.1).At the T c ,the majority of the wild-type amplicons remain double-stranded;however,mutant amplicons are largely dena-tured at the T c and function as template for primer and750Clinical Chemistry 55:4(2009)probe binding.Lowering the temperature from the T c to the annealing and extension temperature allows the probe to bind with the complementary mutant strand. Accordingly,COLD-PCR not only enriches the mutant but also reduces the chance that the probe will mismatch-bind to the wild-type strand by keeping the wild type double-stranded.During the annealing and extension step,the5Ј33Јexonuclease activity of Taq polymerase digests the probe to release the reporter from the quencher,allowing the fluorescence signal to be read at this step(29).The presence and quantity of mutations are detected by recording the threshold cy-cle of the real-time reaction relative to that of refer-ence samples containing known amounts of the same mutation.IMPROVEMENT OF TaqMan GENOTYPING VIA REAL-TIME QUANTITATIVE COLD-PCRValidation of COLD-PCR/TaqMan genotyping was done by means of serial dilutions of DNA from tumor-derived cell lines containing the gefitinib-resistance mutation[CϾT at codon790of EGFR exon20(cell line H1975)](30)or TP53hot-spot mutation GϾA at codon273of TP53exon8(cell line SW480)(31). These T m-reducing mutants are suitable for enrich-ment via fast COLD-PCR.Fig.2depicts representative results comparing the selectivities of regular PCR/ TaqMan genotyping with COLD-PCR/TaqMan geno-typing for a104-bp amplicon from EGFR exon20.The selectivity limit of regular PCR/TaqMan genotyping is about12%mutant allele(Fig.2A);in contrast,COLD-PCR improves the selectivity to0.8%(Fig.2B).Next, we tested whether COLD-PCR/TaqMan genotyping of EGFR exon20encoding the T790M variant can quan-tify the low population of T790M mutations in NSCLC cell lines.Because the EGFR gene is frequently ampli-fied in NSCLC cells,the potential variation in copy number for EGFR exon20needs to be considered be-fore T790M quantification.We applied regular PCR in the presence of the LCGreen dye to quantify the copy numbers for the3NSCLC lines(H820,H3255GR,and PC9GR;Fig.3,A and B).H3255GR exhibits16-fold amplification,and H820and PC9GR exhibit4-fold amplification.On the basis of this quantification,we diluted the genomic DNA from these3cell lines to obtain equal copy numbers of exon20and tested for T790M mutants and known dilutions of T790M mu-tants added to wild-type DNA(Fig.3,C and D).The percentage of T790M was calculated by interpolation to be5%for H820,2.85%for H3255GR,and0.4%for PC9GR.Fig.4,A and B,presents the results of applying real-time COLD-PCR to TaqMan genotyping of the hot-spot mutations in codon273of TP53exon8. Whereas the limit of selectivity for regular PCR/Taq-Man genotyping is about10%mutant allele,COLD-PCR/TaqMan genotyping can detect as little as0.33% mutant alleles among wild-type alleles,an improve-Real-Time COLD-PCRClinical Chemistry55:4(2009)751ment of approximately30-fold.Examination of4can-cer samples,one of which(CT20)is known to contain a codon273GϾA mutation at a low level(approxi-mately5%)(6,7),with COLD-PCR/TaqMan genotyp-ing clearly identified the mutation-containing sample, but regular PCR/TaqMan genotyping did not(Fig.4,C and D).Thus,our data demonstrate that COLD-PCR im-proves TaqMan-genotyping selectivity by15-to30-fold.To understand further the improvement in muta-tion selectivity produced by the application of COLD-PCR,we subjected the PCR product used in TaqMan genotyping of the T790EGFR exon20mutation to an RFLP assay with a restriction enzyme,Nla III,that se-lectively recognizes mutation-containing DNA.The digested products were then examined via denaturing HPLC,as reported previously(22).For comparison to752Clinical Chemistry55:4(2009)COLD-PCR/TaqMan genotyping,we conducted iden-tical experiments after regular PCR/TaqMan genotyp-ing.Fig.5demonstrates that the product produced by regular PCR/TaqMan genotyping and digested with Nla III barely shows the mutant peak(12%mutant rel-ative to wild type),in agreement with the real-time PCR results(Fig.2).In contrast,Nla III-digested prod-ucts produced by COLD-PCR/TaqMan genotyping depict mutant peaks down to0.8%mutant alleles. The data in Fig.5are additional verification that the improved real-time PCR quantification of T790M mu-tations indeed reflects the anticipated mutation-specific products and not false-positive signals. FURTHER IMPROVEMENT OF TaqMan GENOTYPING VIA2 ROUNDS OF COLD-PCR AMPLIFICATIONGiven that a single round of COLD-PCR/TaqMan genotyping can detect as little as0.8%mutant alleles, we tested whether nested COLD-PCR/TaqMan geno-typing can further improve the selectivity of T790M mutant detection.The nested PCR generates a67-bp product from EGFR exon20.When applied directly to genomic DNA(i.e.,not in a nested format),COLD-PCR/TaqMan genotyping of the67-bp region had a selectivity of approximately0.8%T790M mutant al-leles(see Fig.1in the Data Supplement that accompa-nies the online version of this article at http://www. /content/vol55/issue4).When2COLD-PCR TaqMan reactions are applied in series(the first COLD-PCR for a104-bp amplicon and the second a nested PCR for the67-bp amplicon),the combined selectivity for T790M detection is far superior to the selectivity of a single reaction.Fig.6A shows that a sin-gle round of COLD-PCR/TaqMan genotyping fails to detect0.1%T790M mutant alleles.In contrast,2 rounds of COLD-PCR/TaqMan genotyping improved the selectivity to better than0.05%mutant alleles, whereas11replicates of the wild-type DNA remained at the baseline.Thus,2rounds of COLD-PCR com-bined with TaqMan genotyping improve the mutation detection over that obtained with a single round. DiscussionWe have described COLD-PCR/TaqMan genotyping,a real-time mutation-detection methodology that com-Real-Time COLD-PCRClinical Chemistry55:4(2009)753bines COLD-PCR and TaqMan genotyping for detecting the EGFR-encoded T790M mutant and TP53codon273 mutations in serial dilutions of mutant DNA,in cell lines, and in biological samples.The clinical relevance of these mutations is well established.T790M,an acquired muta-tion in the EGFR protein that renders NSCLC patients resistant to gefitinib or erlotinib,is found in approxi-mately50%of tumors from patients who have acquired resistance to these kinase inhibitors(32).The presence of hot-spot mutation at codon273of TP53is a factor for a poor prognosis in NSCLC patients(27).The new method is based on the ability of fast COLD-PCR to enrich T m-reducing mutations and the ability of the Taq-Man probe to detect mutations in a real-time,quantita-tive format.Consequently,a single round of COLD-PCR/ TaqMangenotypingquantitativelydetectsaslittleas0.8% mutant alleles with a15-to30-fold better selectivity than regular PCR/TaqMan genotyping.The addition of a sec-ond round of COLD-PCR/TaqMan genotyping further improves the selectivity and reproducibly identifies1mu-tant allele among2000wild-type alleles.Alternative TaqMan-based approaches that detect low amounts of mutant alleles have been described. Allele-specific PCR-based TaqMan genotyping,Taq-MAMA,uses a mutant-matched nucleotide at the3Јend of a primer and a penultimate3Јmismatch to achieve specific allele discrimination in the PCR(33);however, the optimization of Taq MAMA conditions can be tedious (33).PNA-based TaqMan genotyping uses a PNA to in-hibit wild-type DNA and a mutant-specific TaqMan probe to detect mutations(34).The necessity to define experimental conditions such as probe concentration while retaining not only the compatibility between the PNA probe and the TaqMan probe but also the ability of the PNA to inhibit the wild type increases the complexity of assay development.Scorpion assays(35)provide a good alternative to TaqMan genotyping in that the probe and primer are combined on a single oligonucleotide. DxS Ltd.offers a commercially available combination of Scorpion and ARMS®(amplification refractory mutation system)technologies that can detect low-level mutations such as T790M in EGFR with a sensitivity similar to that of754Clinical Chemistry55:4(2009)the single-round COLD-PCR/TaqMan assay;however,the Scorpion assay is relatively more complex,expensive, and slow(1h for the COLD-PCR/Taqman assay vs2–3h for the Scorpion assay)(35).COLD-PCR achieves real-time mutation detection without tedious optimization or the use of costly PNA probes or Scorpion primers,be-cause COLD-PCR/TaqMan genotyping uses only tem-perature to inhibit amplification of the wild type.An-other potential advantage of the COLD-PCR/TaqMan approach is in the multiplex detection of mutations.Mul-tiplexing would be more difficult to achieve with combi-nations of PNA and TaqMan probes because of the num-ber of oligonucleotides used in the reaction.In summary,without relying on special probes and reagents,COLD-PCR/TaqMan genotyping is sim-ple,fast,easy to use,and low in cost compared with other TaqMan-based mutation-detection methods. The major improvement in selectivity obtained with COLD-PCR enables the popular TaqMan genotyping method to become a powerful tool for detecting low-level mutations in clinical samples.Author Contributions:All authors confirmed they have contributed to the intellectual content of this paper and have met the following3re-quirements:(a)significant contributions to the conception and design, acquisition of data,or analysis and interpretation of data;(b)drafting or revising the article for intellectual content;and(c)final approval of the published article.Authors’Disclosures of Potential Conflicts of Interest:Upon manuscript submission,all authors completed the Disclosures of Poten-tial Conflict of Interest form.Potential conflicts of interest: Employment or Leadership:None declared.Consultant or Advisory Role:P.A.Ja¨nne,AstraZeneca,Roche, Boehringer Ingelheim,and AVEO Pharmaceuticals.Stock Ownership:None declared.Honoraria:None declared.Research Funding:P.A.Ja¨nne,Pfizer;G.M.Makrigiorgos,NIH grants CA-115439and CA-111994;J.Li,NIH training grant5T32 CA09078.Expert Testimony:None declared.Role of Sponsor:The funding organizations played no role in the design of study,choice of enrolled patients,review and interpretation of data,or preparation or approval of manuscript.Real-Time COLD-PCRClinical Chemistry55:4(2009)755References1.Croce CM.Oncogenes and cancer.N Engl J Med2008;358:502–11.2.Bayley H.Sequencing single molecules of DNA.Curr Opin Chem Biol2006;10:628–37.3.Marsh S.Pyrosequencing applications.MethodsMol Biol2007;373:15–24.4.Jenkins GJ,Chaleshtori MH,Song H,Parry JM.Mutation analysis using the restriction site muta-tion(RSM)assay.Mutat Res1998;405:209–20.5.Ragoussis J,Elvidge GP,Kaur K,Colella S.Matrix-assisted laser desorption/ionisation,time-of-flight mass spectrometry in genomics research.PLoS Genet2006;2:e100.6.Li J,Berbeco R,Distel RJ,Ja¨nne PA,Wang L,Makrigiorgos GM.s-RT-MELT for rapid mutation scanning using 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