Read about some of the most commonly asked questions when performing PCR prior to HRM analysis.
Size of the amplicon
Q1. How big or small should the amplicon be?
A: It is typically recommended to use small fragments, about 70-350 bp in size, however, PCR products up to 449 bp have been reported in some studies. Studies with Mycoplasma species also used large fragments over 400 bp in size. Larger amplicons can be analyzed successfully, but usually with lower resolution. This is simply because a single base variation affects the melting behavior of a 100 bp amplicon more than a 500 bp amplicon, for example. Subtle variations, such as class IV SNPs, typically require smaller amplicons for detection. For SNP analysis, the recommended amplicon size is between 70 and 150 bp.
Q2. Is it true that the smaller the amplicon the better the results?
A: This is not always the case because it depends on the sequence surrounding the position that needs to be analyzed. When performing screening, the aim is to get the greatest Tm
difference between homozygotes. If the amplicon size is too small, lower fluorescence signals may be generated. This can be due to a reduced level of dye incorporation in the shorter sequence, making analysis more difficult. In addition, general guidelines should be followed for primer design. PCR products < 70 bp in size should be avoided because Tm
values of primer dimers and the specific amplicon might be very similar.
Specificity of amplification and template purity
Q1. What other factors have an impact on melting?
A: DNA melting behavior is affected by salts in the reaction mix, so it is important that the concentration of buffer, Mg2+
, and other salts is as uniform as possible in all samples. It is recommended to use the same genomic DNA purification procedure for all samples subjected to HRM analysis. This avoids introduction of variations due to differing compositions of elution buffers used in different extraction methods. Home-brew DNA purification methods can inadvertently introduce contaminants and inhibitors. Contaminants such as NaCl can increase the Tm
, while others such as isopropanol can decrease the Tm
. To avoid any reduction in performance, we recommend using QIAGEN genomic DNA purification kits such as QIAamp or DNeasy Kits.
Amount of DNA to use
Q1. How much DNA should be used per reaction?
A: Use 1–50 ng of template genomic DNA or 1–50 pg microbial DNA per 25 μl reaction. We recommend using comparable amounts of template genomic DNA for all samples resulting in CT
(threshold cycle) values below 30 and differing by no more than three CT
values. Control DNA and sample DNA should be of comparable integrity. For example, if analyzing samples from FFPE tissues, control DNA should also be derived from FFPE tissues with comparable integrity.
Q2. What happens if I use too little template for PCR?
A: The capture and analysis of real-time amplification data can be extremely useful when troubleshooting HRM analyses. Amplification plots should have a CT
value of no more than 30. Using too little starting template or template degradation can result in products that amplify later than this and have a CT
value typically >30, which will produce variable HRM results due to amplification artifacts. Therefore, use sufficient pre-amplification template. For DNA from eukaryotic sources, 1–50 ng DNA/reaction is recommended. For DNA from microbial sources, 1–50 pg DNA/reaction is recommended.
Q3. Does DNA concentration matter?
A: DNA quality is closely linked with concentration. Therefore, the DNA purification method must be standardized. QIAGEN offers a wide range of DNA purification products to meet these requirements. DNA samples used for HRM should be normalized in concentration. All DNA samples should be quantified and then adjusted to the same concentration using the same dilution buffer. The concentration of a DNA fragment can affect its melting temperature (Tm
). For this reason, sample DNA concentrations must be kept as similar as possible. Use sufficient PCR cycles so that all samples have reached the plateau phase of PCR to ensure that comparable amounts of PCR product are generated. At plateau, all reactions will have amplified to a similar extent irrespective of their starting amount. Note however that "poor" reactions might not reach plateau with the same amplified quantity due to, for example, inconsistent assay setup (e.g., the primer concentration was too low).
Specificity of PCR
Q1. How can PCR specificity be ensured?
A: Use the Type-it HRM PCR Kit and the EpiTect HRM PCR Kit to generate highly specific PCR products for HRM analysis. Design suitable primers for HRM PCR. Check the concentration, storage conditions, and quality of the template and control DNA, as well as primers. Efficient removal of PCR inhibitors is essential for optimal results. Purify nucleic acids from your sample using an appropriate purification method, such as QIAGEN's QIAamp or DNeasy Kits for genomic DNA purification. Ensure that all reagents, buffers, and solutions used for isolating and dilution of template nucleic acids are free of nucleases (RNases/DNases).
Q2. How does amplification specificity influence HRM?
A: It is critical to ensure that a single, pure PCR product is analyzed. Samples containing post-amplification artifacts such as primer dimers or nonspecific products can make HRM results difficult to interpret. Use the Type-it HRM PCR Kit and the EpiTect HRM PCR Kit to generate highly specific PCR products for HRM analysis. Perform standard melting curve analysis before HRM.
Get the answers to some of the most commonly asked questions when performing post-PCR analysis, prior to setting up your HRM experiment.
Q1. What should I check prior to starting HRM?
A: Check for aberrant amplification plots. Prior to HRM, examine real-time plot data carefully for abnormal amplification curve shapes. Plots having a log-linear phase that is not steep, is jagged, or that reaches a low signal plateau compared to other reactions can indicate poor amplification or a fluorescence signal that is simply too low. Poor reactions can be caused by reaction inhibitors, too little dye, incorrect reaction setup, etc. HRM data from such samples can be inconclusive or of lower resolution.
Q2. Do all the samples used for HRM have to be of the same volume and amount?
A: Yes, it is important to ensure sample-to-sample uniformity. Ensure that comparable amounts and volumes of DNA are used in all samples, noting that all samples should not differ by more than three CT
values. The concentration of a DNA fragment affects its melting temperature (Tm
). For this reason, sample DNA concentrations must be kept as similar as possible. When analyzing amplification products, ensure every reaction has amplified to the plateau phase. At plateau, all reactions will have amplified to a similar extent irrespective of their starting amount. Note however that "poor" reactions might not reach plateau with the same amplified quantity due to, for example, inconsistent assay setup (e.g., the primer concentration was too low). All samples should contain the same concentration of dye. When using the Type-it HRM PCR Kit or the EpiTect HRM PCR Kit for HRM analysis, these requirements are completely fulfilled.
Find out what your HRM results mean and how you can improve them.
Q1. Why is there variability in signal (CT
in HRM) between replicates or samples?
A: Ensure that comparable amounts of DNA are used in all samples, noting that all samples should not differ by more than three CT
values. Note that rare or new genetic variants may generate results outside of the expected ranges. Bubbles in the wells can also result in variation. Spin down plates to remove air bubbles and remove any liquid from the plate cover, and then repeat the post-PCR plate read. Ensure all reaction components are properly mixed.
Q2. Why are the amplification curves not very steep and CT
values higher than expected?
A: This may be caused by PCR inhibition due to the presence of inhibiting agents such as salts and alcohols. These can be derived from DNA purification methods and can influence the Tm
. A single melt peak indicates the presence of a specific PCR product and absence of PCR artifacts or byproducts such as primer dimers. Use QIAGEN DNA purification products to achieve high-quality template DNA.
Q3. Why can I see extra melt curves/greater spread in the same genotype sample observed?
A: When examining mixtures of methylated and unmethylated DNA samples or when an amplicon has a complex composition comprising of different domains that melt at different temperatures, a single, distinct peak is not observed. QIAGEN HRM kits result in melt profiles that are smoother, more tightly grouped, and easier to separate into clear clusters, making analysis straightforward and easy to interpret.
Q4. Why can I see complex melt curves with multiple melt domains?
A: This can be caused by multiple melt regions due to presence of more than one mutation site. Very long sequences or sequences with a biased distribution of GC- and AT-rich sequence stretches can also contain multiple melt regions. Use small fragments 70–350 bp in size.
Q5. How can HRM results be improved?
A: Using standardized kits such as the Type-it HRM PCR Kit and the EpiTect HRM PCR Kit eliminates most variables, ensuring reliable results. In addition, it is recommended to assess:
Data analysis and interpretation
- CT values and integrity of your DNA
- PCR efficiency
- The derivative plot melt curves to determine the presence of a single product and the absence of primer dimers and other PCR artifacts
- Amplification efficiency (using comparative quantitation) to determine the individual kinetics of the reaction
Discover what your HRM data means.
Q1. Are there any in silico programs to detect the expected Tm
A: Poland is a simple program where a wild-type sequence is entered and the Tm
is predicted. The sequence of the changed variant is then entered to generate an expected Tm
. The expected melting temperatures between the two are then compared. MeltSim is another statistical-mechanical program for calculating melting curves (derivative profiles) and maps of DNA.
Q2. How do you know where to place the normalization regions and assign a confidence percentage?
A: You must empirically evaluate this with known controls.
Q3. How can data interpretation be made easier?
A: Allow sufficient data collection for pre- and post-melt phases. Capture HRM data points over about a 10°C (or greater) window, centered around the observed Tm
. This provides enough baseline data points for effective curve normalization and will result in tighter replicates and easier data interpretation.
Q4. What are the software requirements for HRM analysis and data interpretation?
A: HRM data analysis discriminates between genotypes by comparing the position and shape of melting curves of different samples. Standard HRM software packages enable comparison of melt curve shapes and positions with a control. This method can cause unreliable, difficult-to-interpret results, and time-consuming manual data interpretation may be necessary. In contrast, Rotor-Gene ScreenClust HRM Software uses innovative mathematical algorithms to characterize samples and group them into clusters. These methods enable identification of even difficult class IV A/T SNPs, which can have differences in melting temperatures as low as 0.1°C.
HRM technology applications
Find out what HRM can do for your research.
Q1. Can HRM be used for microsatellite analysis?
A: There are some published studies that show microsatellite HRM analysis data. It generally depends on the number of expected permutations and combinations, as HRM is a comparative analysis method and all variables and controls must be in place for an auto call.
Q2. Can multiplexing be performed using HRM?
A: Yes, to a limited extent. If amplicons melt at different temperatures, distinct melting profiles can be observed. HRM analysis of up to 4 small amplicons has been achieved.
Q3. Can predictive tests be performed using a normal melt and derivative plot?
A: This is only applicable to certain changes with large Tm
differences and in certain amplicons. However, these are limiting as an A/T change in class IV SNPs cannot be detected using normal melt curve analysis, but is readily detected using HRM.