High resolution melting analysis (HRM) is a sensitive and rapid solution for genetic variant detection. It depends on sequence differences that result in heteroduplexes changing the shape of the melting curve. By combing HRM and agarose gel electrophoresis, different types of genetic variants such as indels can be identified.
High resolution melting analysis (HRM) is a powerful method for genotyping and genetic variation scanning. Most HRM applications depend on saturating DNA dyes that detect sequence differences, and heteroduplexes that change the shape of the melting curve. Excellent instrument resolution and special data analysis software are needed to identify the small melting curve differences that identify a variant or genotype. Different types of genetic variants with diverse frequencies can be observed in the gene specific for patients with a specific disease, especially cancer and in the CALR gene in patients with Philadelphia chromosome–negative myeloproliferative neoplasms. Single nucleotide changes, insertions and/or deletions (indels) in the gene of interest can be detected by the HRM analysis. The identification of different types of genetic variants is mostly based on the controls used in the qPCR HRM assay. However, as the product length increases, the difference between wild-type and heterozygote curves becomes smaller, and the type of genetic variant is more difficult to determine. Therefore, where indels are the prevalent genetic variant expected in the gene of interest, an additional method such as agarose gel electrophoresis can be used for the clarification of the HRM result. In some instances, an inconclusive result must be re-checked/re-diagnosed by standard Sanger sequencing. In this retrospective study, we applied the method to JAK2 V617F-negative patients with MPN.
Somatic genetic variants in the calreticulin gene (CALR) were recognized in 2013 in patients with myeloproliferative neoplasms (MPN) such as essential thrombocythemia and primary myelofibrosis1,2. Since then, more than 50 genetic variants in the CALR gene have been discovered, inducing a +1 (−1+2) frameshift3. The two most frequent CALR genetic variants are a 52 bp deletion (NM_004343.3 (CALR):c.1099_1150del52, p.(Leu367Thrfs*46)), also called type 1 mutation, and a 5 bp insertion (NM_004343.3 (CALR):c.1154_1155insTTGTC, p.(Lys385Asnfs*47)), also called type 2 mutation. These two genetic variants represent 80% of all CALR genetic variants. The other ones have been classified as type 1–like or type 2–like using algorithms based on the preservation of an α helix close to wild type CALR4. Here, we present one of the highly sensitive and rapid methods for CALR genetic variant detection, the high resolution melting analysis method (HRM). This method enables the rapid detection of type 1 and type 2 genetic variants, which represent the majority of CALR mutations5. HRM was introduced in combination with real time »polymerase chain reaction« (qPCR) in 1997 as a tool to detect the mutation in factor V Leiden6. In comparison to Sanger sequencing that represents the golden standard technique, HRM is a more sensitive and less specific method5. The HRM method is a good screening method that enables a rapid analysis of a large number of samples with a great cost-benefit5. It is a simple PCR method performed in the presence of a fluorescent dye and does not require specific skills. Another benefit is that the procedure itself does not damage or destroy the analyzed sample that allows us to reuse the sample for electrophoresis or Sanger sequencing after the HRM procedure7. The only disadvantage is that it is sometimes difficult to interpret the results. Additionally, HRM does not detect the exact mutation in patients with non-type 1 or type 2 mutations8. In these patients, Sanger sequencing should be performed (Figure 1).
HRM is based on the amplification of the specific DNA region in the presence of saturating DNA fluorescent dye, which is incorporated in double-stranded DNA (dsDNA). The fluorescent dye emits light when incorporated in the dsDNA. After a progressive increase in temperature, dsDNA breaks down into single stranded DNA, which can be detected on the melting curve as a sudden decrease in fluorescence intensity. The shape of the melting curve depends on the DNA sequence that is used to detect the mutation. Melting curves of samples are compared to melting curves of known mutations or wild type CALR. Distinct melting curves represent a different mutation that is non type 1 or type 29.
The algorithm for the somatic genetic variant detection in the CALR gene by HRM, agarose gel electrophoresis and sequencing method (Figure 1) was used and validated in the retrospective study published before10.
The study was approved by the Committee of Medical Ethics of the Republic of Slovenia. All procedures were in accordance with the Helsinki declaration.
1. Fluorescence-based quantitative real-time PCR (qPCR) and post-qPCR analysis by HRM
2. Agarose gel electrophoresis
The successfully amplified DNA region of interest with an exponential increase in fluorescence that exceeds the threshold between cycles 15 and 35 and very narrow values of the cycle of quantification (Cq) in all replicated samples and controls (Figure 2) is a prerequisite for the reliable identification of genetic variants by HRM analysis. This is achieved by using a precise determination of DNA with fluorescence staining and an equal amount of DNA in the qPCR HRM experiment (see step 1.2). Figure 2 shows the successful amplification of the DNA region of interest where the Cq values of all the samples and controls are in a very narrow interval. There is no amplification in the NTC wells.
The HRM stage is performed immediately after qPCR using the protocol described in step 1.11. The active melt regions (Figure 3, label (c)) of the samples, the controls and the NTC are used to create their aligned melt curve plots (Figure 4). Therefore, the correctly set pre- and post-melt regions/temperature lines (Figure 3A) are important for properly visualizing and identifying genetic variants in the samples. Figure 4A and Figure 4B show the aligned melt curves and difference plots, respectively, where the identification of genetic variants is possible. The unknown samples are tightly aligned with one of the positive controls. Figure 3B shows incorrectly set pre- and post-melt regions/temperature lines. This results in an aligned melt curve and difference plots where correct identification of the genetic variants is more difficult (Figure 4C and Figure 4D).
To confirm the HRM results and to detect whether standard or next generation sequencing method is required to identify the genetic variant present in the sample, agarose gel electrophoresis is used. Figure 7 shows the agarose gel electrophoresis of the same samples and controls that are displayed in Figure 4. The genetic variant in the sample can be identified by comparing the band pattern of the sample to the controls and by combining the HRM and agarose gel electrophoresis. However, the correct genetic variant identification can only be done for the samples that contain the same genetic variant as one of the controls used in the HRM assay (Figure 7). Samples containing rare CALR genetic variants differ in the HRM result and electrophoresis band pattern (Figure 8). In this case, the Sanger sequencing or even next generation sequencing method are used to identify the exact genetic variant.
Figure 1: Schematic representation of the algorithm for the somatic genetic variant detection in the CALR gene by HRM, agarose gel electrophoresis and sequencing methods. Please click here to view a larger version of this figure.
Figure 2: Amplification plot of the qPCR HRM assay for the detection of the genetic variants in the CALR gene. The amplification plot is displayed as the raw fluorescence normalized to the fluorescence from the passive reference (ΔRn) and as a function of a cycle number. The baseline is set from 3 to 15 cycles when the DNA region of interest was efficiently amplified using 20 ng of high-quality DNA in the qPCR reaction. The normal amplification plots of the DNA region of interest of all the samples are shown as green lines. Plots show an exponential increase in fluorescence that exceeds the threshold between cycles 15 and 35 in the experiment using 20 ng of high-quality DNA in qPCR reaction. The graph shows no amplification in the non-template sample wells (NTC). Please click here to view a larger version of this figure.
Figure 3: Derivative melt curves in the qPCR HRM assay for the detection of the genetic variants in the CALR gene. A) An example of correctly set pre- and post-melt regions/temperature lines. The pre-melt stop temperature line must be adjacent to the start of the melt transition region. The post-melt start temperature line must be adjacent to the end of the melt transition region. The (a) label indicates the pair of lines to the left of the peaks where the pre-melt starts and stop temperatures are set. Every amplicon is double-stranded. The (b) label indicates the data peaks of the active melt region used to create the aligned melt curves plot. The (c) label indicates the pair of lines to the right of the peaks where the post-melt start and stop temperatures are set. Every amplicon is single-stranded. B) An example of incorrectly set pre- and post-melt regions/temperature lines. The start and stop of the pre- and post-melt temperature lines are not adjacent to the melt transition regions and are more than 0.5 °C apart from each other. Please click here to view a larger version of this figure.
Figure 4: The aligned melt curves and difference plots in the qPCR HRM assay for the detection of the genetic variants in the CALR gene. A) and B) show the aligned melt curves and difference plots after the pre- and post-melt regions/temperature lines are correctly set. A) The aligned melt curves of the positive controles with 52 bp deletion (type 1 mutation), 5 bp insertion (type 2 mutation) and a wild-type are shown as orange, purple and red color, respectively. B) The Difference plot of the same samples as described in panel A. C) and D) show the aligned melt curves and difference plots after the incorrectly set pre- and post-melt regions/temperature lines for the same set of samples. Please click here to view a larger version of this figure.
Figure 5: The gel electrophoresis system. A) The basic units of the gel electrophoresis system (see Table of Materials). B) Gel imager with photo-documentation system consisting of a CCD camera, a chamber with suitable trans/illuminating lights, and a photographic filter is shown (see Table of Materials). Please click here to view a larger version of this figure.
Figure 6: Loading the gel with diluted samples and DNA size standard solution or RNase and DNase free H2O for empty wells. Please click here to view a larger version of this figure.
Figure 7: Analyses of the qPCR HRM products on gel electrophoresis. The gel was exposed to UV transilluminator. Image was taken by the photo-documentation system (Figure 5B). Wells from 1 to 3 show controls: 52 bp deletion (type 1 mutation), 5 bp insertion (type 2 mutation) and wild-type variant, respectively. The band pattern of the unknown samples in the wells 4-10 indicates them as the wild-type genetic variant. The M well represents diluted DNA size standard solution (100 to 2,000 bp). Please click here to view a larger version of this figure.
Figure 8: HRM and gel electrophoresis analyses of the genetic variants in the CALR gene. A) HRM analyses. The aligned melt curves of the positive controls with 52 bp deletion (type 1 mutation), 5 bp insertion (type 2 mutation) and a wild-type are shown as orange, purple and red color, respectively. The unknown sample with the different genetic variant is indicated as dark blue color. B) Gel electrophoresis analyses. Wells from 1 to 3 show controls: 52 bp deletion (type 1 mutation), 5 bp insertion (type 2 mutation) and wild-type variant, respectively. The band pattern of the unknown sample in the lane 9 indicates the different genetic variant as controls. Other unknown samples are the wild-type variant. The M well represents the diluted DNA size standard solution (100 to 2,000 bp). Please click here to view a larger version of this figure.
Figure 9: The limit of detection of HRM assay. A serial dilution of a sample of a 52 bp deletion (type 1 mutation) and 5 bp insertion (type 2 mutation) is presented displaying a genetic variant allele burden of approximately 50% according to Sanger sequencing analysis and the qPCR HRM assay according to the protocol described in this article. A) and B) show the aligned melt curves and difference plots for wild-type and serial dilutions of a 52 bp deletion (type 1 mutation). C) and D) show the aligned melt curves and difference plots for wild-type and serial dilutions of a 5 bp insertion (type 2 mutation). In either case, the CALR genetic variant could be detected in up to 1.56% dilution. Please click here to view a larger version of this figure.
High-resolution melting of DNA is a simple solution for genotyping and genetic variant scanning14. It depends on sequence differences that result in heteroduplexes that change the shape of the melting curve. Different types of genetic variants with diverse frequencies can be observed in the gene specific for a certain group of patients with cancer1,2,15,16,10. Deletions or insertions in the gene of interest can be detected by the HRM analysis as we have shown in Figure 4A. At the implementation of the qPCR HRM assay into the routine testing, we have performed an initial evaluation study including 21 JAK2 V617F-negative ET patients with a median age of 63 years (range from 24 to 90 years). A genetic variant in the CALR gene was detected in 12 out of 21 JAK2 V617F-negative ET patients (proportion 0.57). A 52 bp deletion (type 1 mutation) was detected in 9 out of 21 (proportion 0.43), a 5 bp insertion (type 2 mutation) was detected in 3 out of 21 (proportion 0.143) JAK2 V617F-negative ET patients. The results are consistent with the data from the literature1,2,17.
Reliable identification of genetic variants by HRM analysis can be achieved through the proper assay development that ensures that the experiment is optimized for HRM. Factors other than sequence can be a source of small differences in the HRM curves such as primer design, amplicon length, dye selection, choice of qPCR HRM reagent and temperature and time profiles of the qPCR HRM steps. This is the part of the very early development and optimization of the qPCR HRM assay and has already been discussed elsewhere12,14,18. However, reliable identification of genetic variants in the CALR gene with comparable sensitivity of the assays could be achieved on different HRM platforms using the same primer sequences19. The most critical point in the optimization process of the qPCR HRM assay was optimization of the melting and elongation temperature in the qPCR step of the qPCR HRM assay. No modification was needed for the HRM step12. In the routine testing, other factors influencing the HRM results becomes more important to follow.
High-quality DNA resuspended in a low-salt buffer such as TE (10 mM Tris, 1 mM EDTA or lower concentration) and the same amount of DNA in the qPCR HRM assay are important factors for successfully amplified DNA region of interests with a high signal plateau in the qPCR amplification phase (Figure 2). This leads to reliable identification of genetic variants by HRM analysis (Figure 4A,4B)12,14. A high-salt buffer used for the elution of DNA in the extraction protocol may subtly change the thermodynamics of the DNA melting transition. Precipitation and resuspension in low-salt buffer TE or dilution of the sample can resolve the problem of the impurities in the DNA sample. Low quality DNA can produce nonspecific PCR products or failed amplification. All this can result in a lower reproducibility and higher error rate in HRM variant detection. Additional optimization for the challenging samples such as DNA extracted from paraffin-embedded samples could be needed to obtain an optimal HRM result15.
Large differences in the DNA amount used in the qPCR HRM assay can impact the resulting melting temperature (Tm) and consequently lead to inconclusive results. Therefore, the precise determination of DNA concentration and dilution of all DNA samples are mandatory20. Ultraviolet (UV) absorption and fluorescence staining methods accurately determine the concentrations of high-purity DNA solutions21. The UV absorbance method could be used for evaluating the purity of the DNA solutions by performing ratio absorbance measurements at A260/A280 and A260/230. However, the fluorescent staining method is more sensitive to the degradation of DNA than the UV absorption method and can more accurately determine DNA concentration21. Therefore, it is more appropriate for the standardization of quantification and dilution of all DNA samples in the HRM analysis20,21.
The indels are the main genetic variants in the CALR gene observed in MPN patients1. As the product length changes and increases, the difference between wild-type and heterozygote curves becomes smaller, and the variant detection becomes harder7. Therefore, an additional method for the clarification of such genetic variant should be used.
A good example is agarose gel electrophoresis. This is a simple and effective method to separate DNA fragments of different sizes22,23. DNA molecules are separated by size within the agarose gel so that the traveled distance is inversely proportional to the logarithm of its molecular weight24. Figure 4A, Figure 4B and Figure 7 show examples where the two most common types of CALR genetic variant, 52 bp deletion and 5 bp insertion, are identified by combining qPCR HRM and agarose gel electrophoresis results. However, when the HRM result and agarose gel electrophoresis band pattern indicate a different genetic variant (including single nucleotide change) as in the controls (Figure 8), Sanger sequencing is still needed to identify the exact genetic variant10.
A low level of somatic genetic variant in the CALR gene can be present in the patient's sample making an interpretation of the qPCR HRM, agarose gel electrophoresis and Sanger sequencing results more demanding, particularly at the detection limit of the assay (Figure 9). Any melting curve shape line differing from the wild type one indicates the genetic variant to be present in the sample. The sensitivity of the qPCR HRM assay is lower than 5% (Figure 9 and reference19) and is more sensitive than the Sanger sequencing method, whose sensitivity was reported to be 15-20%19. In these cases, the next generation deep sequencing method that detect larger indels could be applied and confirm the HRM result. In conclusion, the HRM analysis is a powerful method for genotyping and genetic variation scanning of somatic genetic variants in the CALR gene. The identification of different types of genetic variant is mostly based on the controls used in the qPCR HRM assay. More reliable results are obtained by combining the HRM results with results from agarose gel electrophoresis. In case of inconclusive results, standard Sanger sequencing or even more sensitive next generation sequencing method can be used to properly identify the genetic variant.
The authors have nothing to disclose.
The authors would like to thank all the academic experts and employees at the Specialized Hematology Laboratory, Department of Hematology, Division of Internal Medicine, University Medical Centre Ljubljana.
E-Gel EX 4% Agarose | Invitrogen, Thermo Fischer Scientific | G401004 | |
Fuorometer 3.0 QUBIT | Invitrogen, Thermo Fischer Scientific | Q33216 | |
Invitrogen E-Gel iBase and E-Gel Safe Imager Combo Kit | Invitrogen, Thermo Fischer Scientific | G6465EU | |
MeltDoctor HRM MasterMix 2X | Applied Biosystem, Thermo Fischer Scientific | 4415440 | Components: AmpliTaqGold 360 DNA Polymerase, MeltDoctor trade HRM dye, dNTP blend including dUTP, Magnesium salts and other buffer components, precisely formulated to obtain optimal HRM results |
MicroAmp Fast 96-well Reaction Plate (0.1 mL) | Applied Biosystems, Thermo Fischer Scientific | 4346907 | |
MicroAmp Optical adhesive film | Applied Biosystems, Thermo Fischer Scientific | 4311971 | |
NuGenius | Syngene | NG-1045 | Gel documentation systems |
Primer CALRex9 Forward | Eurofins Genomics | Sequence: 5'GGCAAGGCCCTGAGGTGT'3 (High-Purity, Salt-Free) | |
Primer CALRex9 Reverse | Eurofins Genomics | Sequence: 5'GGCCTCAGTCCAGCCCTG'3 (High-Purity, Salt-Free) | |
QIAamp DNA Mini Kit | QIAGEN | 51306 | DNA isolation kit with the buffer for DNA dilution. |
Qubit Assay Tubes | Invitrogen, Thermo Fischer Scientific | Q32856 | |
QUBIT dsDNA HS assay | Invitrogen, Thermo Fischer Scientific | Q32854 | |
Trackit 100bp DNA Ladder | Invitrogen, Thermo Fischer Scientific | 10488058 | Ladder consists of 13 individual fragments with the reference bands at 2000, 1500, and 600 bp. |
ViiA7 Real-Time PCR System | Applied Biosystems, Thermo Fischer Scientific | 4453534 | |
Water nuclease free | VWR, Life Science | 436912C | RNase, DNase and Protease free water |