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Listed below are some common symptoms in an HRM experiment. Select one of the symptoms to view possible causes and solutions.
Related Topics: Gene Expression/Quantification Experiments and Allelic Discrimination Experiments.
Missed call or clustering
Amplification did not reach plateau
Poor amplification or failed reaction
A missed call or clustering may be due to the causes below. Select one of the causes for possible solutions.
Small thermal changes between two sequences can be difficult to differentiate, and these changes can arise from sources such as sequence differences, sample quality, amplicon length, primer design, and PCR reagents.
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In some cases, the GC content of the primers can affect the melting temperature of your target. Ensuring that the temperature shift will not be affected by the sequence of your primer set will improve calls and clustering.
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An insufficient amplification where your reactions do not reach plateau may be due to the causes below. Select one of the causes for possible solutions.
Low-quality DNA may prevent samples from reaching high plateau, which can result in inconclusive high resolution melt (HRM) data. Improving the DNA quality will ensure that the HRM data is not skewed and will be called appropriately.
The length of an amplicon can influence the amplification and melt profile if the sequence contains more than one mutation. As a result, the mutation of interest can be difficult to separate from the other sequence variants if primer sets are designed for longer amplicons (> 300 bp).
Poor amplification or failed reactions may be due to the causes below. Select one of the causes for possible solutions.
Successful HRM analysis is highly dependent on the quality of the amplicons being compared. Low-quality DNA may produce nonspecific PCR products that can affect the melt profile.
Sometimes inhibitors of PCR are carried over from sample preparation (nucleic acid extraction). Common PCR inhibitors include phenol, detergents, proteases, and residual compounds from source materials, such as animal or plant tissue, body fluids, or soil preparations. Suboptimal reactions containing inhibitors are likely to affect the reactions and thus any postanalysis, such as HRM analysis.
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