HHS Public Access Author manuscript Author Manuscript
Clin Chem. Author manuscript; available in PMC 2016 December 14. Published in final edited form as: Clin Chem. 2015 November ; 61(11): 1354–1362. doi:10.1373/clinchem.2015.245357.
DMSO increases mutation-scanning detection sensitivity in clinical samples using high resolution melting Chen Song1, Elena Castellanos-Rizaldos1, Rafael Bejar2, Benjamin L. Ebert3, and G. Mike Makrigiorgos1,* 1Department
of Radiation Oncology, Dana-Farber Cancer Institute, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA
Author Manuscript
2Division
of Hematology and Oncology, UCSD Moores Cancer Center, La Jolla, CA
3Division
of Hematology, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA
Abstract BACKGROUND—Mutation scanning provides the simplest, lowest cost method for identifying DNA variations on single PCR amplicons, and it may be performed prior to sequencing to avoid screening of non-informative wild type samples. High resolution melting (HRM) is the most commonly used method for mutation scanning. However, by using PCR-HRM mutations below ≈ 3–10% that can still be clinically significant may often be missed. Therefore, enhancing HRM detection sensitivity is important for mutation scanning and its clinical application.
Author Manuscript
METHODS—We used serial dilution of TP53 exon 8 mutation containing cell lines to demonstrate the improvement in detection sensitivity for conventional-PCR-HRM in the presence of DMSO. We also conducted full-COLD-PCR to further enrich low-level mutations prior to HRM ±DMSO and employed droplet-digital PCR to derive the optimal conditions for mutation enrichment. Both conventional-PCR-HRM and full-COLD-PCR-HRM ±DMSO were used for mutation scanning in TP53 exon 8 in cancer samples containing known mutations and in myelodysplastic syndrome samples with unknown mutations. Mutations in other genes were also examined.
Author Manuscript
RESULTS—The detection sensitivity of PCR-HRM-scanning increases 2–5-fold in the presence of DMSO, depending also on mutation type and sequence context, and can typically detect mutation abundance of about 1%. When mutation enrichment is applied during amplification using full-COLD-PCR and followed by HRM in the presence of DMSO, mutations with 0.2–0.3% mutation abundance in TP53 exon 8 can be detected. CONCLUSIONS—DMSO improves HRM mutation scanning sensitivity. When full-COLD-PCR is employed, followed by DMSO-HRM, the overall improvement is about 20-fold as compared to conventional PCR-HRM.
*
Correspondence address: G. Mike Makrigiorgos, Ph.D., Brigham and Women’s Hospital, Level L2, Radiation Therapy, 75 Francis Street, Boston, MA 02115, USA., Tel: (+1) 617-525-7122. Fax: (+1) 617-582-6037, [email protected]. CONFLICT OF INTEREST STATEMENT: COLD-PCR is a technology owned by the Dana-Farber Cancer Institute and has been commercially licensed. The manuscript has not been published previously and is not being considered concurrently by another journal. All authors and acknowledged contributors have read and approved the manuscript.
Song et al.
Page 2
Author Manuscript
Keywords High resolution melting; Mutation scanning; DMSO; COLD-PCR; low-level mutation detection
INTRODUCTION
Author Manuscript
Discovery and detection of molecular biomarkers for gene diagnosis has become a fast growing field in the era of precision medicine. Mutation scanning without previous knowledge of the mutation type or position is of importance because it can help screening for multiple clinically important biomarkers in target genes, as well as to discover new biomarkers with potential clinical significance. Sequencing is the gold standard for mutation discovery and identification, and its cost is steadily decreasing as much effort is made to reduce the cost of data generation and variation in data management and to improve the data interpretation in downstream analysis of next-generation sequencing (1–4). Nevertheless, mutation scanning provides the lowest cost method for identifying DNA variations on single PCR amplicons and it may be performed prior to sequencing to avoid screening of noninformative wild type samples when few sequences are to be investigated. Accordingly, methods like high resolution melting (HRM) (5, 6) are commonly used in clinical application for mutation scanning (7, 8). HRM is normally conducted in a post-PCR fashion and can be easily done in a routine laboratory with a real-time PCR machine. However, using PCR-HRM may often miss mutations below about 3–10% (9) that may be clinically significant, such as mutations in plasma. Therefore, increasing detection sensitivity or HRM mutation scanning can increase its clinical value.
Author Manuscript Author Manuscript
Here we examine the influence of adding dimethyl sulfoxide (DMSO), to the HRM detection sensitivity. DMSO additive is used for increasing the amplification efficiency on GC-rich sequences (10, 11), because it enables destabilization of duplex DNA by opening up secondary structures or by weakening hydrogen bonds between base pairs (12, 13). Destabilization of DNA via addition of betaine prior to melting analysis of real time PCR products generates a narrower melting peak for the probe-template duplex (14), while addition of high salt buffer may improve clustering in HRM analysis (15). We thus hypothesized that DMSO would affect the thermal stability of wild type DNA and mutant type DNA to a different degree, enlarging their melting profile difference during HRM analysis, thus increasing the detection sensitivity of HRM. In addition to DMSO we examined concomitant CO-amplification at Lower Denaturation temperature-PCR. COLDPCR (16, 17) is another way to increase HRM detection sensitivity, by providing mutation enrichment during PCR, based on DNA melting properties (18) and with minimal change to existing conventional PCR workflows (19–22). Full-COLD-PCR (16, 19) is a COLD-PCR version able to enrich multiple mutations along a target sequence by including an extra step for cross-hybridization to generate mismatched duplexes followed by preferential denaturation and amplification at optimized critical temperature. We report on the use of DMSO to increase the detection sensitivity of PCR-HRM and the combination with full-COLD-PCR to provide a combined improvement.
Clin Chem. Author manuscript; available in PMC 2016 December 14.
Song et al.
Page 3
Author Manuscript
MATERIAL AND METHODS Cell lines and clinical samples Missense mutations in several positions of the TP53 exon 8 were assessed in this study. Human genomic DNA from commercial cell lines SW480 (ATCC no. CCL-228™, p.R273H, c.818G>A), HCC1008 (ATCC no. CRL-2320™, p.D281H, c.841G>C), PFSK-1 (ATCC no. CRL-2060™, p.C275G, c.823T>G) was extracted using the DNeasy™ Blood and Tissue kit (Qiagen, Valencia, CA, USA) following the manufacturer’s protocol. Human genomic DNA (Promega Corporation, Madison, WI, USA) was used as wild type control DNA and for creating mixtures of wild type and mutant type DNA with gradually decreasing mutation abundances. Standard reference DNA containing multiple low-abundance mutations in a variety of genes was purchased from Horizon Diagnostics (Cambridge, UK).
Author Manuscript
To evaluate the utility of the present approach in characterizing specimens from different origins we analyzed whole genome-amplified DNA from eleven myelodysplastic syndrome (MDS) samples without detectable TP53 exon 8 mutation when screened via MALDI-TOF or conventional PCR coupled with Sanger sequencing (23, 24). We also evaluated genomic DNA from three lung tumor samples (TL6, TL8 and TL121) that previously confirmed to harbor missense mutations present at a low frequency (2%, 1% and 1% respectively), and a colorectal tumor sample CT20 with a missense mutation at an abundance of ~1% (22, 25) (Supplementary Table 1a). Conventional PCR and full-COLD-PCR
Author Manuscript Author Manuscript
PCR reactions were performed on SmartCycler real-time PCR system (Cepheid, Sunnyvale, CA, USA) or CFX Connect™ real-time PCR (Bio-Rad Labratories, Hercules, CA, USA) using Phusion High-Fidelity DNA polymerase (New England Biolabs, Ipswich, MA, USA) and LCGreen Plus+ as a fluorescence dye (BioFire Diagnositics, Salt Lake City, UT, USA). Primers were designed for TP53 exon 8 and five other genes (Supplementary Table 1b). Ten ng genomic DNA were used as template in a 25 μL PCR mix including 1X Phusion HF buffer, 200 nM of each primer, 200 μM of each of the four dNTPs, 0.8 X LCGreen and 0.5 unit of Phusion polymerase. For conventional PCR, an initial denaturation step was performed for 2 min at 98°C, followed by 45 cycles of 10 sec denaturation at 98°C, 20 sec annealing at 58°C and 10 sec elongation at 72°C. The final step included a melting curve (0.2°C step increments), 2 or 4 seconds hold before each acquisition) from 65 to 95°C. For full-COLD-PCR, initial optimization was conducted to establish the main factors that affect the enrichment efficiency. As shown in supplementary materials, programs were conducted to find out the optimal combination of conventional PCR cycle number n and full-COLDPCR cycle number m, and the critical temperature (Tc) for mutant-preferential amplification. Then the program for full-COLD-PCR was set with an initial denaturation step at 98°C for 2 min, followed by n cycles of conventional PCR cycling (98°C, 10 sec; 58°C, 20 sec; 72°C, 10 sec) and m cycles of full-COLD-PCR cycling (98°C, 10 sec; 72°C, 30 sec; Tc, 10 sec; 58°C, 20 sec; 72°C, 10 sec), such that m+n = 45, and with a final melting curve from 65°C to 95°C.
Clin Chem. Author manuscript; available in PMC 2016 December 14.
Song et al.
Page 4
High resolution melting (HRM) analysis with DMSO
Author Manuscript
Ten-microliter of each conventional PCR product or full-COLD-PCR product was transferred to a 96-well plate, and DMSO was added into each sample and mixed well to make a final concentration of 5%, 7% or 10%. Finally, 20 μl of mineral oil was added to each well. Each sample was also evaluated without adding DMSO for HRM on the same plate as a control sample. HRM was performed on a 96-well LightScanner® system (Idaho Technology, Salt Lake City, UT, USA). The software sensitivity level was set as 1.2 for computing DNA variant groups. All experiments were replicated at least three independent times for assessing the reproducibility of the results. Droplet-digital PCR for mutation abundance validation
Author Manuscript
Droplet-digital PCR (ddPCR) reactions were carried out as previously described (26), to verify the mutation abundance of cell line DNA samples. Sequences of primers and probes are in Supplementary Table 1b. Amplifications were performed in a 20 μl volume containing 1x ddPCR Supermix for probes (Bio-Rad), 900 nM forward and reverse primers (synthesized by Integrated DNA Technologies Inc. Coralville, IA, USA), 250 nM FAM and HEX probes (Integrated DNA Technologies) and 10 ng genomic DNA or real-time PCR products (using 1 to 5,000,000 final dilution for conventional PCR or full-COLD-PCR products). Droplets were then generated using the DG8™ droplet generator cartridges (BioRad) which contained 20 μl aqueous phase with 70 μl of droplet generation (DG) oil (BioRad). Samples were transferred to a 96-well reaction plate and then sealed using the PX1 PCR plate sealer (Bio-Rad) for 10 s at 180°C prior to thermal cycling. The thermal cycling program was performed on an Eppendorf Mastercycler ep Gradient S (Eppendorf, Hamburg, Germany) with an initial denaturation step at 95°C for 10 min, followed by 40 cycles of 30 sec denaturation at 94°C, 60 sec annealing at 58°C, and with a final step holding at 98°C for 10 min. Then the plate was transferred to QX100 droplet reader (Bio-Rad) for endpoint reading. Calculation of absolute number of positive events for a given channel (FAM or HEX), the ratio and the fractional abundance of mutation for each sample were performed by the Quantasoft Software (Bio-Rad). The determination of number of target copies per droplet (number of copies of target molecule) was adjusted by the software to fit a Poisson distribution model with 95% confidence level.
Author Manuscript
Sanger sequencing
Author Manuscript
The PCR products were digested by Exonuclease I and Shrimp Alkaline Phosphatase (New England Biolabs) and processed for Sanger sequencing at Eton Bioscience Inc. (Boston, MA, USA). To enable sequencing of short PCR amplicons, a 30-T tail was added to the 5′end of the forward primer (Supplementary Table 1b).
RESULTS DMSO effect on improving HRM detection sensitivity In order to examine the effect of DMSO on HRM scanning, we conducted conventional PCR from serially diluted cell line DNA that contained 3 different mutations in TP53 exon 8, into WT control DNA. The mutation abundances obtained by dilution were verified with ddPCR
Clin Chem. Author manuscript; available in PMC 2016 December 14.
Song et al.
Page 5
Author Manuscript
prior to amplification, as shown in Figure 1. Then varying amounts of DMSO, 5–10%, were added to PCR products prior to HRM analysis. Compared to PCR-HRM scanning without DMSO, increasing DMSO concentration to 5% and 7% improved the mutation discrimination, Figure 2. A 7% DMSO enables discrimination of three different mutations down to 1% mutation abundance (Figure 2), and improves the detection sensitivity limits of conventional HRM analysis (~3–10% abundance, (9)). Increasing the DMSO concentration to 10% didn’t improve further the HRM detection sensitivity, while created more variations in the melting profiles (not shown). Accordingly, we used 7% DMSO for post-PCR products in HRM scanning for subsequent experiments.
Author Manuscript
To examine whether sequences other than TP53 exon 8 display improved sensitivity upon post-PCR addition of 7% DMSO, we diluted DNA with mutations in five additional genes, BRAF, FLT3, IDH1, NRAS and JAK2 into WT DNA such that, a 1% mutation abundance was obtained. Supplementary Figure 1S panel A demonstrates that in all but one gene this method was able to detect down to 1% mutation level. In the case of JAK2 the improvement was not identified as significant by the software. Finally we examined whether addition of DMSO prior to PCR also allows the same improvement in detection sensitivity. Supplementary Figure 1S panel B indicates a similar level of HRM sensitivity with postPCR addition of DMSO. Full-COLD-PCR optimization and HRM analysis
Author Manuscript
(a) Primer design—There are two main steps central to implementing full-COLD-PCR. These are the cross-hybridization of template DNA strands to create hetero-duplexes between mutated and WT DNA strands, by lowering the temperature after complete denaturation; and a subsequent preferential denaturation step, during which the duplex strands potentially containing mismatches denature first, resulting in preferential amplification of mutant DNA (16). The temperature for DNA strand cross-hybridization should be such that does not allow primers to bind during this step. Accordingly, primer melting temperatures often have to be adjusted to avoid unwanted template replication during cross-hybridization. A control experiment in order to ensure that primers do not bind during the temperature used for cross-hybridization, entails a separate, two-stage conventional PCR. Supplementary Figure S2 panel A shows that, at an annealing temperature of 72°C the two-stage conventional PCR generates no PCR product for any annealing time. Accordingly, this combination of temperature/primers is deemed appropriate for full-COLD-PCR since primers may not bind the template during the cross-hybridization step.
Author Manuscript
(b) Optimization of conventional PCR cycles before switching to full-COLDPCR cycling—When amplifying directly from genomic DNA, a set of conventional PCR cycles precedes full COLD-PCR in order to build enough template for efficient crosshybridization (16). DNA hybridization rate is proportional to its concentration, hence hybridization is faster when there are more DNA molecules in the solution. We conducted a series of PCR reactions on DNA samples containing 0.6% mutation c.841G>C (p.D281H) in TP53 exon 8, with different combination of “n” cycles of conventional PCR and “m” cycles of full-COLD PCR, while keeping the sum of “n” and “m” constant, 45. The resulting
Clin Chem. Author manuscript; available in PMC 2016 December 14.
Song et al.
Page 6
Author Manuscript
mutation abundance was evaluated by applying ddPCR before and after reactions. Supplementary Figure S2B shows the effect of n and m on the final mutation enrichment. When n is close to the PCR threshold value (Ct), the mutation enrichment is highest. A combination of 25 cycles conventional PCR and 20 cycles of full-COLD-PCR was chosen for further work. Additionally, with other conditions remaining the same, extension of heteroduplex hybridizing time from 30 seconds to 2 minutes didn’t improve substantially the enrichment efficiency (data not shown); therefore, 30 seconds was chosen for further work.
Author Manuscript
(c) Optimal critical denaturation temperature on two PCR machines—Another major factor affecting mutation enrichment during full-COLD-PCR is the critical temperature to enable the preferential denaturation of the mismatched heteroduplexes. We applied a gradient of critical temperatures to examine its effect on the mutation enrichment (Supplementary Figure S2C). The data show a substantial enrichment in a temperature window about 0.8°C (89.1–89.9°C) for Tm decreasing and Tm retaining mutations (SW480, p.R273H, c.818 G>A and HCC1008, p.R273H, c.841G>C). And the Tm increasing mutation (PFSK-1, p.C275G, c.823 T>G) shows lower enrichment efficiency and narrower critical temperature window around 0.4°C (89.3–89.7°C). Therefore, we chose 89.5°C as a single critical temperature for full-COLD-PCR cycling to ensure substantial enrichment for all possible mutations in TP53 exon 8. Finally, to test the differences in critical temperatures for full-COLD-PCR conducted on a different PCR machine, we performed the same test on a Bio-Rad CFX 96-well real-time PCR system. The same tendency among different mutation types was demonstrated (Supplementary Figure S2D), although the optimal temperature range shifted by 0.5°C to higher temperatures on Bio-Rad PCR relative to Cepheid SmartCycler. 90.1°C was chosen as critical temperature for full-COLD-PCR cycling on the Bio-Rad PCR system.
Author Manuscript
HRM in the presence of post-full-COLD-PCR DMSO Using optimized conditions for full-COLD-PCR, we tested HRM mutation scanning using serial dilution of DNA containing 3 different TP53 exon 8 mutations, into WT DNA. FullCOLD-PCR increased HRM detection sensitivity for all three mutations down to 1–2% mutation abundance (Figure 3A–C). The mutation enrichment was confirmed via ddPCR (Supplementary Table 2). In the presence of 7% DMSO, full-COLD-PCR products showed improved detection sensitivity with HRM scanning for all the three cell lines (Figure 3D–F). Mutation abundances of 0.2–0.3% was discriminated from WT DNA via HRM after fullCOLD-PCR in the presence of 7% DMSO (Supplementary Figure S3), producing an overall increase in HRM detection sensitivity by ~20-fold, as compared to conventional PCR-HRM without DMSO.
Author Manuscript
Mutation-screening of clinical samples We applied both conventional-PCR-HRM and the optimized full-COLD-PCR-HRM protocol to eleven MDS samples with unknown mutation status, plus three lung tumor samples and one colorectal sample previously shown to contain TP53 exon 8 mutations at low abundance (19). When conventional-PCR-HRM without DMSO was applied, only one sample (MDS sample SM-2DD63) was discriminated from wild type samples (Figure 4A). When 7% DMSO was added during HRM, two more samples (MDS sample SM-2DD7X Clin Chem. Author manuscript; available in PMC 2016 December 14.
Song et al.
Page 7
Author Manuscript
and colorectal sample CT20) were distinguished from wild type samples (Figure 4B). Application of full-COLD-PCR-HRM with or without DMSO was able to identify mutations in one additional MDS sample (SM-2DD6R) than the DMSO enhanced conventional-PCRHRM, plus all the lung tumor and colorectal samples that were previously confirmed to have mutations in TP53 exon 8 (Figure 4C–D). By performing Sanger sequencing on the fullCOLD-PCR amplified samples with mutations, the type and position of the MDS samples de-novo-discovered mutations, revealed via COLD-PCR-HRM-DMSO, was identified. SM-2DD63 sample revealed a c.818 G>A mutation and SM-2DD7X sample revealed a c. 844 C>T mutation (Supplementary Figure S4).
DISCUSSION
Author Manuscript
Our data demonstrate that the detection limits in HRM analysis, a commonly used process for mutation scanning in clinical and research applications, can be improved by simply adding DMSO to increase the thermo-dynamic difference between wild type and mutant type DNA. Conventional PCR followed by HRM with 7% DMSO was able to detect mutation abundance of about 1%, and if this is preceded by full-COLD-PCR the detection sensitivity improves further and mutation abundances down to 0.2–0.3% can be discriminated from wild type. This improves the ability for detection of low level mutations in clinical samples. Adding DMSO to PCR-HRM analysis revealed one additional mutation in an MDS sample, which was previously below the PCR-HRM mutation detection levels. Recent work has demonstrated that such mutations can have independent prognostic significance in MDS (24) and detection of clonal as well as low-level mutations in hematologic malignancies is becoming clinically important (27, 28).
Author Manuscript
The intrinsic property of full-COLD-PCR for enriching multiple mutations in the target amplicons makes it a most suitable platform for closed tube mutation scanning in combination with HRM. While more sensitive forms of COLD-PCR have been described (29–31) these are not easily combined with HRM and examine shorter DNA sequences as compared to full-COLD-PCR. Full-COLD-PCR does not require a change in instrumentation and in most cases can retain the originally used PCR primers. However, the optimization workflow for selection of primers and critical denaturation temperatures described herein (Supplementary Figure 2 Frames A and C) must be followed and established for each amplicon.
Author Manuscript
Addition of DMSO to improve HRM analysis can be performed either before or after PCR and can be applied to any platform based on HRM detection. Among HRM applications, HRM genotyping (5) is more frequently used as compared to HRM mutation scanning. Since the DMSO effect on DNA duplex stability should be general, it may be possible that DMSO can also improve the detection sensitivity in HRM genotyping. It is also possible that organic solvents such as betaine and formamide that are used as PCR modifiers (14, 32–35) may improve HRM analysis.
Supplementary Material Refer to Web version on PubMed Central for supplementary material.
Clin Chem. Author manuscript; available in PMC 2016 December 14.
Song et al.
Page 8
Author Manuscript
Acknowledgments This work was supported by National Cancer Institute grant number R21CA-175542 (GMM). The contents of this manuscript do not necessarily represent the official views of the National Cancer Institute or the National Institutes of Health.
References
Author Manuscript Author Manuscript Author Manuscript
1. Desai AN, Jere A. Next-generation sequencing: Ready for the clinics? Clin Genet. 2012; 81:503–10. [PubMed: 22375550] 2. Pabinger S, Dander A, Fischer M, Snajder R, Sperk M, Efremova M, et al. A survey of tools for variant analysis of next-generation genome sequencing data. Brief Bioinform. 2014; 15:256–78. [PubMed: 23341494] 3. Robasky K, Lewis NE, Church GM. The role of replicates for error mitigation in next-generation sequencing. Nat Rev Genet. 2014; 15:56–62. [PubMed: 24322726] 4. Sboner A, Mu XJ, Greenbaum D, Auerbach RK, Gerstein MB. The real cost of sequencing: Higher than you think! Genome Biol. 2011; 12:125. [PubMed: 21867570] 5. Zhou L, Wang L, Palais R, Pryor R, Wittwer CT. High-resolution DNA melting analysis for simultaneous mutation scanning and genotyping in solution. Clin Chem. 2005; 51:1770–7. [PubMed: 16189378] 6. Erali M, Wittwer CT. High resolution melting analysis for gene scanning. Methods. 2010; 50:250– 61. [PubMed: 20085814] 7. Kennerson ML, Warburton T, Nelis E, Brewer M, Polly P, De Jonghe P, et al. Mutation scanning the gjb1 gene with high-resolution melting analysis: Implications for mutation scanning of genes for charcot-marie-tooth disease. Clin Chem. 2007; 53:349–52. [PubMed: 17200131] 8. Margraf RL, Mao R, Highsmith WE, Holtegaard LM, Wittwer CT. Mutation scanning of the ret protooncogene using high-resolution melting analysis. Clin Chem. 2006; 52:138–41. [PubMed: 16391329] 9. Ney JT, Froehner S, Roesler A, Buettner R, Merkelbach-Bruse S. High-resolution melting analysis as a sensitive prescreening diagnostic tool to detect kras, braf, pik3ca, and akt1 mutations in formalin-fixed, paraffin-embedded tissues. Archives of pathology & laboratory medicine. 2012; 136:983–92. [PubMed: 22938585] 10. Jensen MA, Fukushima M, Davis RW. Dmso and betaine greatly improve amplification of gc-rich constructs in de novo synthesis. PLoS One. 2010; 5:e11024. [PubMed: 20552011] 11. Varadaraj K, Skinner DM. Denaturants or cosolvents improve the specificity of pcr amplification of a g + c-rich DNA using genetically engineered DNA polymerases. Gene. 1994; 140:1–5. [PubMed: 8125324] 12. Chester N, Marshak DR. Dimethyl sulfoxide-mediated primer tm reduction: A method for analyzing the role of renaturation temperature in the polymerase chain reaction. Anal Biochem. 1993; 209:284–90. [PubMed: 8470801] 13. Escara JF, Hutton JR. Thermal stability and renaturation of DNA in dimethyl sulfoxide solutions: Acceleration of the renaturation rate. Biopolymers. 1980; 19:1315–27. [PubMed: 7397315] 14. Luo T, Jiang L, Sun W, Fu G, Mei J, Gao Q. Multiplex real-time pcr melting curve assay to detect drug-resistant mutations of mycobacterium tuberculosis. J Clin Microbiol. 2011; 49:3132–8. [PubMed: 21752982] 15. Vossen RH, Aten E, Roos A, den Dunnen JT. High-resolution melting analysis (hrma): More than just sequence variant screening. Hum Mutat. 2009; 30:860–6. [PubMed: 19418555] 16. Li J, Wang L, Mamon H, Kulke MH, Berbeco R, Makrigiorgos GM. Replacing pcr with cold-pcr enriches variant DNA sequences and redefines the sensitivity of genetic testing. Nat Med. 2008; 14:579–84. [PubMed: 18408729] 17. Li J, Makrigiorgos GM. Cold-pcr: A new platform for highly improved mutation detection in cancer and genetic testing. Biochem Soc Trans. 2009; 37:427–32. [PubMed: 19290875]
Clin Chem. Author manuscript; available in PMC 2016 December 14.
Song et al.
Page 9
Author Manuscript Author Manuscript Author Manuscript Author Manuscript
18. Milbury CA, Li J, Makrigiorgos GM. Cold-pcr-enhanced high-resolution melting enables rapid and selective identification of low-level unknown mutations. Clin Chem. 2009; 55:2130–43. [PubMed: 19815609] 19. Li J, Milbury CA, Li C, Makrigiorgos GM. Two-round coamplification at lower denaturation temperature-pcr (cold-pcr)-based sanger sequencing identifies a novel spectrum of low-level mutations in lung adenocarcinoma. Hum Mutat. 2009; 30:1583–90. [PubMed: 19760750] 20. Pritchard CC, Akagi L, Reddy PL, Joseph L, Tait JF. Cold-pcr enhanced melting curve analysis improves diagnostic accuracy for kras mutations in colorectal carcinoma. BMC Clin Pathol. 2010; 10:6. [PubMed: 21110880] 21. Mancini I, Santucci C, Sestini R, Simi L, Pratesi N, Cianchi F, et al. The use of cold-pcr and highresolution melting analysis improves the limit of detection of kras and braf mutations in colorectal cancer. J Mol Diagn. 2010; 12:705–11. [PubMed: 20616366] 22. Castellanos-Rizaldos E, Liu P, Milbury CA, Guha M, Brisci A, Cremonesi L, et al. Temperaturetolerant cold-pcr reduces temperature stringency and enables robust mutation enrichment. Clin Chem. 2012; 58:1130–8. [PubMed: 22587896] 23. Bejar R, Levine R, Ebert BL. Unraveling the molecular pathophysiology of myelodysplastic syndromes. J Clin Oncol. 2011; 29:504–15. [PubMed: 21220588] 24. Murphy DM, Bejar R, Stevenson K, Neuberg D, Shi Y, Cubrich C, et al. Nras mutations with low allele burden have independent prognostic significance for patients with lower risk myelodysplastic syndromes. Leukemia. 2013; 27:2077–81. [PubMed: 23708912] 25. Guha M, Castellanos-Rizaldos E, Liu P, Mamon H, Makrigiorgos GM. Differential strand separation at critical temperature: A minimally disruptive enrichment method for low-abundance unknown DNA mutations. Nucleic acids research. 2013; 41:e50. [PubMed: 23258702] 26. Castellanos-Rizaldos E, Paweletz C, Song C, Oxnard GR, Mamon H, Janne PA, Makrigiorgos GM. Enhanced ratio of signals enables digital mutation scanning for rare allele detection. J Mol Diagn. 2015; 17:284–92. [PubMed: 25772705] 27. Wong TN, Ramsingh G, Young AL, Miller CA, Touma W, Welch JS, et al. Role of tp53 mutations in the origin and evolution of therapy-related acute myeloid leukaemia. Nature. 2015; 518:552–5. [PubMed: 25487151] 28. Jaiswal S, Fontanillas P, Flannick J, Manning A, Grauman PV, Mar BG, et al. Age-related clonal hematopoiesis associated with adverse outcomes. The New England journal of medicine. 2014; 371:2488–98. [PubMed: 25426837] 29. Milbury CA, Li J, Makrigiorgos GM. Ice-cold-pcr enables rapid amplification and robust enrichment for low-abundance unknown DNA mutations. Nucleic acids research. 2011; 39:e2. [PubMed: 20937629] 30. How Kit A, Mazaleyrat N, Daunay A, Nielsen HM, Terris B, Tost J. Sensitive detection of kras mutations using enhanced-ice-cold-pcr mutation enrichment and direct sequence identification. Hum Mutat. 2013; 34:1568–80. [PubMed: 24038839] 31. Milbury CA, Correll M, Quackenbush J, Rubio R, Makrigiorgos GM. Cold-pcr enrichment of rare cancer mutations prior to targeted amplicon resequencing. Clin Chem. 2012; 58:580–9. [PubMed: 22194627] 32. Chakrabarti R, Schutt CE. The enhancement of pcr amplification by low molecular weight amides. Nucleic acids research. 2001; 29:2377–81. [PubMed: 11376156] 33. Sarkar G, Kapelner S, Sommer SS. Formamide can dramatically improve the specificity of pcr. Nucleic acids research. 1990; 18:7465. [PubMed: 2259646] 34. Henke W, Herdel K, Jung K, Schnorr D, Loening SA. Betaine improves the pcr amplification of gc-rich DNA sequences. Nucleic acids research. 1997; 25:3957–8. [PubMed: 9380524] 35. Weissensteiner T, Lanchbury JS. Strategy for controlling preferential amplification and avoiding false negatives in pcr typing. Biotechniques. 1996; 21:1102–8. [PubMed: 8969839]
Clin Chem. Author manuscript; available in PMC 2016 December 14.
Song et al.
Page 10
Author Manuscript Author Manuscript
Figure 1.
Work flowchart. The mutation containing genomic DNA is amplified by conventional PCR or full-COLD-PCR, followed by HRM scanning. The enrichment effect before and after full-COLD-PCR is confirmed by ddPCR.
Author Manuscript Author Manuscript Clin Chem. Author manuscript; available in PMC 2016 December 14.
Song et al.
Page 11
Author Manuscript Author Manuscript Author Manuscript
Figure 2.
Conventional PCR products from cell line DNA for HRM scanning (A–C) and post-PCR DMSO effect on the HRM analysis in the presence of 5% (D-F) and 7% DMSO (G–I).
Author Manuscript Clin Chem. Author manuscript; available in PMC 2016 December 14.
Song et al.
Page 12
Author Manuscript Author Manuscript Author Manuscript
Figure 3.
Full-COLD-PCR products from cell line DNA for HRM scanning. (A–C) HRM detection without DMSO; (D–F) HRM detection in the presence of 7% DMSO. Three TP53 exon 8 mutations were tested, HCC1008, p.R273H, c.841G>C; SW480, p.R273H, c.818 G>A; PFSK-1, p.C275G, c.823 T>G.
Author Manuscript Clin Chem. Author manuscript; available in PMC 2016 December 14.
Song et al.
Page 13
Author Manuscript Author Manuscript Figure 4.
Author Manuscript
Conventional PCR products and full-COLD-PCR products of colorectal and lung cancer tumor DNA samples and MDS DNA samples screened via HRM. post-PCR DMSO effect on the HRM analysis is shown. (A) conventional PCR-HRM without DMSO; (B) conventional PCR-HRM with 7% DMSO; (C) full-COLD-PCR-HRM without DMSO; (D) full-COLD-PCR-HRM with 7% DMSO
Author Manuscript Clin Chem. Author manuscript; available in PMC 2016 December 14.
Clin Chem. Author manuscript; available in PMC 2016 December 14. Published in final edited form as: Clin Chem. 2015 November ; 61(11): 1354–1362. doi:10.1373/clinchem.2015.245357.
DMSO increases mutation-scanning detection sensitivity in clinical samples using high resolution melting Chen Song1, Elena Castellanos-Rizaldos1, Rafael Bejar2, Benjamin L. Ebert3, and G. Mike Makrigiorgos1,* 1Department
of Radiation Oncology, Dana-Farber Cancer Institute, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA
Author Manuscript
2Division
of Hematology and Oncology, UCSD Moores Cancer Center, La Jolla, CA
3Division
of Hematology, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA
Abstract BACKGROUND—Mutation scanning provides the simplest, lowest cost method for identifying DNA variations on single PCR amplicons, and it may be performed prior to sequencing to avoid screening of non-informative wild type samples. High resolution melting (HRM) is the most commonly used method for mutation scanning. However, by using PCR-HRM mutations below ≈ 3–10% that can still be clinically significant may often be missed. Therefore, enhancing HRM detection sensitivity is important for mutation scanning and its clinical application.
Author Manuscript
METHODS—We used serial dilution of TP53 exon 8 mutation containing cell lines to demonstrate the improvement in detection sensitivity for conventional-PCR-HRM in the presence of DMSO. We also conducted full-COLD-PCR to further enrich low-level mutations prior to HRM ±DMSO and employed droplet-digital PCR to derive the optimal conditions for mutation enrichment. Both conventional-PCR-HRM and full-COLD-PCR-HRM ±DMSO were used for mutation scanning in TP53 exon 8 in cancer samples containing known mutations and in myelodysplastic syndrome samples with unknown mutations. Mutations in other genes were also examined.
Author Manuscript
RESULTS—The detection sensitivity of PCR-HRM-scanning increases 2–5-fold in the presence of DMSO, depending also on mutation type and sequence context, and can typically detect mutation abundance of about 1%. When mutation enrichment is applied during amplification using full-COLD-PCR and followed by HRM in the presence of DMSO, mutations with 0.2–0.3% mutation abundance in TP53 exon 8 can be detected. CONCLUSIONS—DMSO improves HRM mutation scanning sensitivity. When full-COLD-PCR is employed, followed by DMSO-HRM, the overall improvement is about 20-fold as compared to conventional PCR-HRM.
*
Correspondence address: G. Mike Makrigiorgos, Ph.D., Brigham and Women’s Hospital, Level L2, Radiation Therapy, 75 Francis Street, Boston, MA 02115, USA., Tel: (+1) 617-525-7122. Fax: (+1) 617-582-6037, [email protected]. CONFLICT OF INTEREST STATEMENT: COLD-PCR is a technology owned by the Dana-Farber Cancer Institute and has been commercially licensed. The manuscript has not been published previously and is not being considered concurrently by another journal. All authors and acknowledged contributors have read and approved the manuscript.
Song et al.
Page 2
Author Manuscript
Keywords High resolution melting; Mutation scanning; DMSO; COLD-PCR; low-level mutation detection
INTRODUCTION
Author Manuscript
Discovery and detection of molecular biomarkers for gene diagnosis has become a fast growing field in the era of precision medicine. Mutation scanning without previous knowledge of the mutation type or position is of importance because it can help screening for multiple clinically important biomarkers in target genes, as well as to discover new biomarkers with potential clinical significance. Sequencing is the gold standard for mutation discovery and identification, and its cost is steadily decreasing as much effort is made to reduce the cost of data generation and variation in data management and to improve the data interpretation in downstream analysis of next-generation sequencing (1–4). Nevertheless, mutation scanning provides the lowest cost method for identifying DNA variations on single PCR amplicons and it may be performed prior to sequencing to avoid screening of noninformative wild type samples when few sequences are to be investigated. Accordingly, methods like high resolution melting (HRM) (5, 6) are commonly used in clinical application for mutation scanning (7, 8). HRM is normally conducted in a post-PCR fashion and can be easily done in a routine laboratory with a real-time PCR machine. However, using PCR-HRM may often miss mutations below about 3–10% (9) that may be clinically significant, such as mutations in plasma. Therefore, increasing detection sensitivity or HRM mutation scanning can increase its clinical value.
Author Manuscript Author Manuscript
Here we examine the influence of adding dimethyl sulfoxide (DMSO), to the HRM detection sensitivity. DMSO additive is used for increasing the amplification efficiency on GC-rich sequences (10, 11), because it enables destabilization of duplex DNA by opening up secondary structures or by weakening hydrogen bonds between base pairs (12, 13). Destabilization of DNA via addition of betaine prior to melting analysis of real time PCR products generates a narrower melting peak for the probe-template duplex (14), while addition of high salt buffer may improve clustering in HRM analysis (15). We thus hypothesized that DMSO would affect the thermal stability of wild type DNA and mutant type DNA to a different degree, enlarging their melting profile difference during HRM analysis, thus increasing the detection sensitivity of HRM. In addition to DMSO we examined concomitant CO-amplification at Lower Denaturation temperature-PCR. COLDPCR (16, 17) is another way to increase HRM detection sensitivity, by providing mutation enrichment during PCR, based on DNA melting properties (18) and with minimal change to existing conventional PCR workflows (19–22). Full-COLD-PCR (16, 19) is a COLD-PCR version able to enrich multiple mutations along a target sequence by including an extra step for cross-hybridization to generate mismatched duplexes followed by preferential denaturation and amplification at optimized critical temperature. We report on the use of DMSO to increase the detection sensitivity of PCR-HRM and the combination with full-COLD-PCR to provide a combined improvement.
Clin Chem. Author manuscript; available in PMC 2016 December 14.
Song et al.
Page 3
Author Manuscript
MATERIAL AND METHODS Cell lines and clinical samples Missense mutations in several positions of the TP53 exon 8 were assessed in this study. Human genomic DNA from commercial cell lines SW480 (ATCC no. CCL-228™, p.R273H, c.818G>A), HCC1008 (ATCC no. CRL-2320™, p.D281H, c.841G>C), PFSK-1 (ATCC no. CRL-2060™, p.C275G, c.823T>G) was extracted using the DNeasy™ Blood and Tissue kit (Qiagen, Valencia, CA, USA) following the manufacturer’s protocol. Human genomic DNA (Promega Corporation, Madison, WI, USA) was used as wild type control DNA and for creating mixtures of wild type and mutant type DNA with gradually decreasing mutation abundances. Standard reference DNA containing multiple low-abundance mutations in a variety of genes was purchased from Horizon Diagnostics (Cambridge, UK).
Author Manuscript
To evaluate the utility of the present approach in characterizing specimens from different origins we analyzed whole genome-amplified DNA from eleven myelodysplastic syndrome (MDS) samples without detectable TP53 exon 8 mutation when screened via MALDI-TOF or conventional PCR coupled with Sanger sequencing (23, 24). We also evaluated genomic DNA from three lung tumor samples (TL6, TL8 and TL121) that previously confirmed to harbor missense mutations present at a low frequency (2%, 1% and 1% respectively), and a colorectal tumor sample CT20 with a missense mutation at an abundance of ~1% (22, 25) (Supplementary Table 1a). Conventional PCR and full-COLD-PCR
Author Manuscript Author Manuscript
PCR reactions were performed on SmartCycler real-time PCR system (Cepheid, Sunnyvale, CA, USA) or CFX Connect™ real-time PCR (Bio-Rad Labratories, Hercules, CA, USA) using Phusion High-Fidelity DNA polymerase (New England Biolabs, Ipswich, MA, USA) and LCGreen Plus+ as a fluorescence dye (BioFire Diagnositics, Salt Lake City, UT, USA). Primers were designed for TP53 exon 8 and five other genes (Supplementary Table 1b). Ten ng genomic DNA were used as template in a 25 μL PCR mix including 1X Phusion HF buffer, 200 nM of each primer, 200 μM of each of the four dNTPs, 0.8 X LCGreen and 0.5 unit of Phusion polymerase. For conventional PCR, an initial denaturation step was performed for 2 min at 98°C, followed by 45 cycles of 10 sec denaturation at 98°C, 20 sec annealing at 58°C and 10 sec elongation at 72°C. The final step included a melting curve (0.2°C step increments), 2 or 4 seconds hold before each acquisition) from 65 to 95°C. For full-COLD-PCR, initial optimization was conducted to establish the main factors that affect the enrichment efficiency. As shown in supplementary materials, programs were conducted to find out the optimal combination of conventional PCR cycle number n and full-COLDPCR cycle number m, and the critical temperature (Tc) for mutant-preferential amplification. Then the program for full-COLD-PCR was set with an initial denaturation step at 98°C for 2 min, followed by n cycles of conventional PCR cycling (98°C, 10 sec; 58°C, 20 sec; 72°C, 10 sec) and m cycles of full-COLD-PCR cycling (98°C, 10 sec; 72°C, 30 sec; Tc, 10 sec; 58°C, 20 sec; 72°C, 10 sec), such that m+n = 45, and with a final melting curve from 65°C to 95°C.
Clin Chem. Author manuscript; available in PMC 2016 December 14.
Song et al.
Page 4
High resolution melting (HRM) analysis with DMSO
Author Manuscript
Ten-microliter of each conventional PCR product or full-COLD-PCR product was transferred to a 96-well plate, and DMSO was added into each sample and mixed well to make a final concentration of 5%, 7% or 10%. Finally, 20 μl of mineral oil was added to each well. Each sample was also evaluated without adding DMSO for HRM on the same plate as a control sample. HRM was performed on a 96-well LightScanner® system (Idaho Technology, Salt Lake City, UT, USA). The software sensitivity level was set as 1.2 for computing DNA variant groups. All experiments were replicated at least three independent times for assessing the reproducibility of the results. Droplet-digital PCR for mutation abundance validation
Author Manuscript
Droplet-digital PCR (ddPCR) reactions were carried out as previously described (26), to verify the mutation abundance of cell line DNA samples. Sequences of primers and probes are in Supplementary Table 1b. Amplifications were performed in a 20 μl volume containing 1x ddPCR Supermix for probes (Bio-Rad), 900 nM forward and reverse primers (synthesized by Integrated DNA Technologies Inc. Coralville, IA, USA), 250 nM FAM and HEX probes (Integrated DNA Technologies) and 10 ng genomic DNA or real-time PCR products (using 1 to 5,000,000 final dilution for conventional PCR or full-COLD-PCR products). Droplets were then generated using the DG8™ droplet generator cartridges (BioRad) which contained 20 μl aqueous phase with 70 μl of droplet generation (DG) oil (BioRad). Samples were transferred to a 96-well reaction plate and then sealed using the PX1 PCR plate sealer (Bio-Rad) for 10 s at 180°C prior to thermal cycling. The thermal cycling program was performed on an Eppendorf Mastercycler ep Gradient S (Eppendorf, Hamburg, Germany) with an initial denaturation step at 95°C for 10 min, followed by 40 cycles of 30 sec denaturation at 94°C, 60 sec annealing at 58°C, and with a final step holding at 98°C for 10 min. Then the plate was transferred to QX100 droplet reader (Bio-Rad) for endpoint reading. Calculation of absolute number of positive events for a given channel (FAM or HEX), the ratio and the fractional abundance of mutation for each sample were performed by the Quantasoft Software (Bio-Rad). The determination of number of target copies per droplet (number of copies of target molecule) was adjusted by the software to fit a Poisson distribution model with 95% confidence level.
Author Manuscript
Sanger sequencing
Author Manuscript
The PCR products were digested by Exonuclease I and Shrimp Alkaline Phosphatase (New England Biolabs) and processed for Sanger sequencing at Eton Bioscience Inc. (Boston, MA, USA). To enable sequencing of short PCR amplicons, a 30-T tail was added to the 5′end of the forward primer (Supplementary Table 1b).
RESULTS DMSO effect on improving HRM detection sensitivity In order to examine the effect of DMSO on HRM scanning, we conducted conventional PCR from serially diluted cell line DNA that contained 3 different mutations in TP53 exon 8, into WT control DNA. The mutation abundances obtained by dilution were verified with ddPCR
Clin Chem. Author manuscript; available in PMC 2016 December 14.
Song et al.
Page 5
Author Manuscript
prior to amplification, as shown in Figure 1. Then varying amounts of DMSO, 5–10%, were added to PCR products prior to HRM analysis. Compared to PCR-HRM scanning without DMSO, increasing DMSO concentration to 5% and 7% improved the mutation discrimination, Figure 2. A 7% DMSO enables discrimination of three different mutations down to 1% mutation abundance (Figure 2), and improves the detection sensitivity limits of conventional HRM analysis (~3–10% abundance, (9)). Increasing the DMSO concentration to 10% didn’t improve further the HRM detection sensitivity, while created more variations in the melting profiles (not shown). Accordingly, we used 7% DMSO for post-PCR products in HRM scanning for subsequent experiments.
Author Manuscript
To examine whether sequences other than TP53 exon 8 display improved sensitivity upon post-PCR addition of 7% DMSO, we diluted DNA with mutations in five additional genes, BRAF, FLT3, IDH1, NRAS and JAK2 into WT DNA such that, a 1% mutation abundance was obtained. Supplementary Figure 1S panel A demonstrates that in all but one gene this method was able to detect down to 1% mutation level. In the case of JAK2 the improvement was not identified as significant by the software. Finally we examined whether addition of DMSO prior to PCR also allows the same improvement in detection sensitivity. Supplementary Figure 1S panel B indicates a similar level of HRM sensitivity with postPCR addition of DMSO. Full-COLD-PCR optimization and HRM analysis
Author Manuscript
(a) Primer design—There are two main steps central to implementing full-COLD-PCR. These are the cross-hybridization of template DNA strands to create hetero-duplexes between mutated and WT DNA strands, by lowering the temperature after complete denaturation; and a subsequent preferential denaturation step, during which the duplex strands potentially containing mismatches denature first, resulting in preferential amplification of mutant DNA (16). The temperature for DNA strand cross-hybridization should be such that does not allow primers to bind during this step. Accordingly, primer melting temperatures often have to be adjusted to avoid unwanted template replication during cross-hybridization. A control experiment in order to ensure that primers do not bind during the temperature used for cross-hybridization, entails a separate, two-stage conventional PCR. Supplementary Figure S2 panel A shows that, at an annealing temperature of 72°C the two-stage conventional PCR generates no PCR product for any annealing time. Accordingly, this combination of temperature/primers is deemed appropriate for full-COLD-PCR since primers may not bind the template during the cross-hybridization step.
Author Manuscript
(b) Optimization of conventional PCR cycles before switching to full-COLDPCR cycling—When amplifying directly from genomic DNA, a set of conventional PCR cycles precedes full COLD-PCR in order to build enough template for efficient crosshybridization (16). DNA hybridization rate is proportional to its concentration, hence hybridization is faster when there are more DNA molecules in the solution. We conducted a series of PCR reactions on DNA samples containing 0.6% mutation c.841G>C (p.D281H) in TP53 exon 8, with different combination of “n” cycles of conventional PCR and “m” cycles of full-COLD PCR, while keeping the sum of “n” and “m” constant, 45. The resulting
Clin Chem. Author manuscript; available in PMC 2016 December 14.
Song et al.
Page 6
Author Manuscript
mutation abundance was evaluated by applying ddPCR before and after reactions. Supplementary Figure S2B shows the effect of n and m on the final mutation enrichment. When n is close to the PCR threshold value (Ct), the mutation enrichment is highest. A combination of 25 cycles conventional PCR and 20 cycles of full-COLD-PCR was chosen for further work. Additionally, with other conditions remaining the same, extension of heteroduplex hybridizing time from 30 seconds to 2 minutes didn’t improve substantially the enrichment efficiency (data not shown); therefore, 30 seconds was chosen for further work.
Author Manuscript
(c) Optimal critical denaturation temperature on two PCR machines—Another major factor affecting mutation enrichment during full-COLD-PCR is the critical temperature to enable the preferential denaturation of the mismatched heteroduplexes. We applied a gradient of critical temperatures to examine its effect on the mutation enrichment (Supplementary Figure S2C). The data show a substantial enrichment in a temperature window about 0.8°C (89.1–89.9°C) for Tm decreasing and Tm retaining mutations (SW480, p.R273H, c.818 G>A and HCC1008, p.R273H, c.841G>C). And the Tm increasing mutation (PFSK-1, p.C275G, c.823 T>G) shows lower enrichment efficiency and narrower critical temperature window around 0.4°C (89.3–89.7°C). Therefore, we chose 89.5°C as a single critical temperature for full-COLD-PCR cycling to ensure substantial enrichment for all possible mutations in TP53 exon 8. Finally, to test the differences in critical temperatures for full-COLD-PCR conducted on a different PCR machine, we performed the same test on a Bio-Rad CFX 96-well real-time PCR system. The same tendency among different mutation types was demonstrated (Supplementary Figure S2D), although the optimal temperature range shifted by 0.5°C to higher temperatures on Bio-Rad PCR relative to Cepheid SmartCycler. 90.1°C was chosen as critical temperature for full-COLD-PCR cycling on the Bio-Rad PCR system.
Author Manuscript
HRM in the presence of post-full-COLD-PCR DMSO Using optimized conditions for full-COLD-PCR, we tested HRM mutation scanning using serial dilution of DNA containing 3 different TP53 exon 8 mutations, into WT DNA. FullCOLD-PCR increased HRM detection sensitivity for all three mutations down to 1–2% mutation abundance (Figure 3A–C). The mutation enrichment was confirmed via ddPCR (Supplementary Table 2). In the presence of 7% DMSO, full-COLD-PCR products showed improved detection sensitivity with HRM scanning for all the three cell lines (Figure 3D–F). Mutation abundances of 0.2–0.3% was discriminated from WT DNA via HRM after fullCOLD-PCR in the presence of 7% DMSO (Supplementary Figure S3), producing an overall increase in HRM detection sensitivity by ~20-fold, as compared to conventional PCR-HRM without DMSO.
Author Manuscript
Mutation-screening of clinical samples We applied both conventional-PCR-HRM and the optimized full-COLD-PCR-HRM protocol to eleven MDS samples with unknown mutation status, plus three lung tumor samples and one colorectal sample previously shown to contain TP53 exon 8 mutations at low abundance (19). When conventional-PCR-HRM without DMSO was applied, only one sample (MDS sample SM-2DD63) was discriminated from wild type samples (Figure 4A). When 7% DMSO was added during HRM, two more samples (MDS sample SM-2DD7X Clin Chem. Author manuscript; available in PMC 2016 December 14.
Song et al.
Page 7
Author Manuscript
and colorectal sample CT20) were distinguished from wild type samples (Figure 4B). Application of full-COLD-PCR-HRM with or without DMSO was able to identify mutations in one additional MDS sample (SM-2DD6R) than the DMSO enhanced conventional-PCRHRM, plus all the lung tumor and colorectal samples that were previously confirmed to have mutations in TP53 exon 8 (Figure 4C–D). By performing Sanger sequencing on the fullCOLD-PCR amplified samples with mutations, the type and position of the MDS samples de-novo-discovered mutations, revealed via COLD-PCR-HRM-DMSO, was identified. SM-2DD63 sample revealed a c.818 G>A mutation and SM-2DD7X sample revealed a c. 844 C>T mutation (Supplementary Figure S4).
DISCUSSION
Author Manuscript
Our data demonstrate that the detection limits in HRM analysis, a commonly used process for mutation scanning in clinical and research applications, can be improved by simply adding DMSO to increase the thermo-dynamic difference between wild type and mutant type DNA. Conventional PCR followed by HRM with 7% DMSO was able to detect mutation abundance of about 1%, and if this is preceded by full-COLD-PCR the detection sensitivity improves further and mutation abundances down to 0.2–0.3% can be discriminated from wild type. This improves the ability for detection of low level mutations in clinical samples. Adding DMSO to PCR-HRM analysis revealed one additional mutation in an MDS sample, which was previously below the PCR-HRM mutation detection levels. Recent work has demonstrated that such mutations can have independent prognostic significance in MDS (24) and detection of clonal as well as low-level mutations in hematologic malignancies is becoming clinically important (27, 28).
Author Manuscript
The intrinsic property of full-COLD-PCR for enriching multiple mutations in the target amplicons makes it a most suitable platform for closed tube mutation scanning in combination with HRM. While more sensitive forms of COLD-PCR have been described (29–31) these are not easily combined with HRM and examine shorter DNA sequences as compared to full-COLD-PCR. Full-COLD-PCR does not require a change in instrumentation and in most cases can retain the originally used PCR primers. However, the optimization workflow for selection of primers and critical denaturation temperatures described herein (Supplementary Figure 2 Frames A and C) must be followed and established for each amplicon.
Author Manuscript
Addition of DMSO to improve HRM analysis can be performed either before or after PCR and can be applied to any platform based on HRM detection. Among HRM applications, HRM genotyping (5) is more frequently used as compared to HRM mutation scanning. Since the DMSO effect on DNA duplex stability should be general, it may be possible that DMSO can also improve the detection sensitivity in HRM genotyping. It is also possible that organic solvents such as betaine and formamide that are used as PCR modifiers (14, 32–35) may improve HRM analysis.
Supplementary Material Refer to Web version on PubMed Central for supplementary material.
Clin Chem. Author manuscript; available in PMC 2016 December 14.
Song et al.
Page 8
Author Manuscript
Acknowledgments This work was supported by National Cancer Institute grant number R21CA-175542 (GMM). The contents of this manuscript do not necessarily represent the official views of the National Cancer Institute or the National Institutes of Health.
References
Author Manuscript Author Manuscript Author Manuscript
1. Desai AN, Jere A. Next-generation sequencing: Ready for the clinics? Clin Genet. 2012; 81:503–10. [PubMed: 22375550] 2. Pabinger S, Dander A, Fischer M, Snajder R, Sperk M, Efremova M, et al. A survey of tools for variant analysis of next-generation genome sequencing data. Brief Bioinform. 2014; 15:256–78. [PubMed: 23341494] 3. Robasky K, Lewis NE, Church GM. The role of replicates for error mitigation in next-generation sequencing. Nat Rev Genet. 2014; 15:56–62. [PubMed: 24322726] 4. Sboner A, Mu XJ, Greenbaum D, Auerbach RK, Gerstein MB. The real cost of sequencing: Higher than you think! Genome Biol. 2011; 12:125. [PubMed: 21867570] 5. Zhou L, Wang L, Palais R, Pryor R, Wittwer CT. High-resolution DNA melting analysis for simultaneous mutation scanning and genotyping in solution. Clin Chem. 2005; 51:1770–7. [PubMed: 16189378] 6. Erali M, Wittwer CT. High resolution melting analysis for gene scanning. Methods. 2010; 50:250– 61. [PubMed: 20085814] 7. Kennerson ML, Warburton T, Nelis E, Brewer M, Polly P, De Jonghe P, et al. Mutation scanning the gjb1 gene with high-resolution melting analysis: Implications for mutation scanning of genes for charcot-marie-tooth disease. Clin Chem. 2007; 53:349–52. [PubMed: 17200131] 8. Margraf RL, Mao R, Highsmith WE, Holtegaard LM, Wittwer CT. Mutation scanning of the ret protooncogene using high-resolution melting analysis. Clin Chem. 2006; 52:138–41. [PubMed: 16391329] 9. Ney JT, Froehner S, Roesler A, Buettner R, Merkelbach-Bruse S. High-resolution melting analysis as a sensitive prescreening diagnostic tool to detect kras, braf, pik3ca, and akt1 mutations in formalin-fixed, paraffin-embedded tissues. Archives of pathology & laboratory medicine. 2012; 136:983–92. [PubMed: 22938585] 10. Jensen MA, Fukushima M, Davis RW. Dmso and betaine greatly improve amplification of gc-rich constructs in de novo synthesis. PLoS One. 2010; 5:e11024. [PubMed: 20552011] 11. Varadaraj K, Skinner DM. Denaturants or cosolvents improve the specificity of pcr amplification of a g + c-rich DNA using genetically engineered DNA polymerases. Gene. 1994; 140:1–5. [PubMed: 8125324] 12. Chester N, Marshak DR. Dimethyl sulfoxide-mediated primer tm reduction: A method for analyzing the role of renaturation temperature in the polymerase chain reaction. Anal Biochem. 1993; 209:284–90. [PubMed: 8470801] 13. Escara JF, Hutton JR. Thermal stability and renaturation of DNA in dimethyl sulfoxide solutions: Acceleration of the renaturation rate. Biopolymers. 1980; 19:1315–27. [PubMed: 7397315] 14. Luo T, Jiang L, Sun W, Fu G, Mei J, Gao Q. Multiplex real-time pcr melting curve assay to detect drug-resistant mutations of mycobacterium tuberculosis. J Clin Microbiol. 2011; 49:3132–8. [PubMed: 21752982] 15. Vossen RH, Aten E, Roos A, den Dunnen JT. High-resolution melting analysis (hrma): More than just sequence variant screening. Hum Mutat. 2009; 30:860–6. [PubMed: 19418555] 16. Li J, Wang L, Mamon H, Kulke MH, Berbeco R, Makrigiorgos GM. Replacing pcr with cold-pcr enriches variant DNA sequences and redefines the sensitivity of genetic testing. Nat Med. 2008; 14:579–84. [PubMed: 18408729] 17. Li J, Makrigiorgos GM. Cold-pcr: A new platform for highly improved mutation detection in cancer and genetic testing. Biochem Soc Trans. 2009; 37:427–32. [PubMed: 19290875]
Clin Chem. Author manuscript; available in PMC 2016 December 14.
Song et al.
Page 9
Author Manuscript Author Manuscript Author Manuscript Author Manuscript
18. Milbury CA, Li J, Makrigiorgos GM. Cold-pcr-enhanced high-resolution melting enables rapid and selective identification of low-level unknown mutations. Clin Chem. 2009; 55:2130–43. [PubMed: 19815609] 19. Li J, Milbury CA, Li C, Makrigiorgos GM. Two-round coamplification at lower denaturation temperature-pcr (cold-pcr)-based sanger sequencing identifies a novel spectrum of low-level mutations in lung adenocarcinoma. Hum Mutat. 2009; 30:1583–90. [PubMed: 19760750] 20. Pritchard CC, Akagi L, Reddy PL, Joseph L, Tait JF. Cold-pcr enhanced melting curve analysis improves diagnostic accuracy for kras mutations in colorectal carcinoma. BMC Clin Pathol. 2010; 10:6. [PubMed: 21110880] 21. Mancini I, Santucci C, Sestini R, Simi L, Pratesi N, Cianchi F, et al. The use of cold-pcr and highresolution melting analysis improves the limit of detection of kras and braf mutations in colorectal cancer. J Mol Diagn. 2010; 12:705–11. [PubMed: 20616366] 22. Castellanos-Rizaldos E, Liu P, Milbury CA, Guha M, Brisci A, Cremonesi L, et al. Temperaturetolerant cold-pcr reduces temperature stringency and enables robust mutation enrichment. Clin Chem. 2012; 58:1130–8. [PubMed: 22587896] 23. Bejar R, Levine R, Ebert BL. Unraveling the molecular pathophysiology of myelodysplastic syndromes. J Clin Oncol. 2011; 29:504–15. [PubMed: 21220588] 24. Murphy DM, Bejar R, Stevenson K, Neuberg D, Shi Y, Cubrich C, et al. Nras mutations with low allele burden have independent prognostic significance for patients with lower risk myelodysplastic syndromes. Leukemia. 2013; 27:2077–81. [PubMed: 23708912] 25. Guha M, Castellanos-Rizaldos E, Liu P, Mamon H, Makrigiorgos GM. Differential strand separation at critical temperature: A minimally disruptive enrichment method for low-abundance unknown DNA mutations. Nucleic acids research. 2013; 41:e50. [PubMed: 23258702] 26. Castellanos-Rizaldos E, Paweletz C, Song C, Oxnard GR, Mamon H, Janne PA, Makrigiorgos GM. Enhanced ratio of signals enables digital mutation scanning for rare allele detection. J Mol Diagn. 2015; 17:284–92. [PubMed: 25772705] 27. Wong TN, Ramsingh G, Young AL, Miller CA, Touma W, Welch JS, et al. Role of tp53 mutations in the origin and evolution of therapy-related acute myeloid leukaemia. Nature. 2015; 518:552–5. [PubMed: 25487151] 28. Jaiswal S, Fontanillas P, Flannick J, Manning A, Grauman PV, Mar BG, et al. Age-related clonal hematopoiesis associated with adverse outcomes. The New England journal of medicine. 2014; 371:2488–98. [PubMed: 25426837] 29. Milbury CA, Li J, Makrigiorgos GM. Ice-cold-pcr enables rapid amplification and robust enrichment for low-abundance unknown DNA mutations. Nucleic acids research. 2011; 39:e2. [PubMed: 20937629] 30. How Kit A, Mazaleyrat N, Daunay A, Nielsen HM, Terris B, Tost J. Sensitive detection of kras mutations using enhanced-ice-cold-pcr mutation enrichment and direct sequence identification. Hum Mutat. 2013; 34:1568–80. [PubMed: 24038839] 31. Milbury CA, Correll M, Quackenbush J, Rubio R, Makrigiorgos GM. Cold-pcr enrichment of rare cancer mutations prior to targeted amplicon resequencing. Clin Chem. 2012; 58:580–9. [PubMed: 22194627] 32. Chakrabarti R, Schutt CE. The enhancement of pcr amplification by low molecular weight amides. Nucleic acids research. 2001; 29:2377–81. [PubMed: 11376156] 33. Sarkar G, Kapelner S, Sommer SS. Formamide can dramatically improve the specificity of pcr. Nucleic acids research. 1990; 18:7465. [PubMed: 2259646] 34. Henke W, Herdel K, Jung K, Schnorr D, Loening SA. Betaine improves the pcr amplification of gc-rich DNA sequences. Nucleic acids research. 1997; 25:3957–8. [PubMed: 9380524] 35. Weissensteiner T, Lanchbury JS. Strategy for controlling preferential amplification and avoiding false negatives in pcr typing. Biotechniques. 1996; 21:1102–8. [PubMed: 8969839]
Clin Chem. Author manuscript; available in PMC 2016 December 14.
Song et al.
Page 10
Author Manuscript Author Manuscript
Figure 1.
Work flowchart. The mutation containing genomic DNA is amplified by conventional PCR or full-COLD-PCR, followed by HRM scanning. The enrichment effect before and after full-COLD-PCR is confirmed by ddPCR.
Author Manuscript Author Manuscript Clin Chem. Author manuscript; available in PMC 2016 December 14.
Song et al.
Page 11
Author Manuscript Author Manuscript Author Manuscript
Figure 2.
Conventional PCR products from cell line DNA for HRM scanning (A–C) and post-PCR DMSO effect on the HRM analysis in the presence of 5% (D-F) and 7% DMSO (G–I).
Author Manuscript Clin Chem. Author manuscript; available in PMC 2016 December 14.
Song et al.
Page 12
Author Manuscript Author Manuscript Author Manuscript
Figure 3.
Full-COLD-PCR products from cell line DNA for HRM scanning. (A–C) HRM detection without DMSO; (D–F) HRM detection in the presence of 7% DMSO. Three TP53 exon 8 mutations were tested, HCC1008, p.R273H, c.841G>C; SW480, p.R273H, c.818 G>A; PFSK-1, p.C275G, c.823 T>G.
Author Manuscript Clin Chem. Author manuscript; available in PMC 2016 December 14.
Song et al.
Page 13
Author Manuscript Author Manuscript Figure 4.
Author Manuscript
Conventional PCR products and full-COLD-PCR products of colorectal and lung cancer tumor DNA samples and MDS DNA samples screened via HRM. post-PCR DMSO effect on the HRM analysis is shown. (A) conventional PCR-HRM without DMSO; (B) conventional PCR-HRM with 7% DMSO; (C) full-COLD-PCR-HRM without DMSO; (D) full-COLD-PCR-HRM with 7% DMSO
Author Manuscript Clin Chem. Author manuscript; available in PMC 2016 December 14.
