Listed below are some common symptoms in an allelic discrimination experiment. Select one of the symptoms to view possible causes and solutions.
Related Topics: Gene Expression/Quantification Experiments and High Resolution Melt (HRM) Experiments.
More than three clusters
Homozygotes as heterozygotes
Inaccurate grouping of your data into more than 3 clusters may be due to the causes below. Select one of the causes for possible solutions.
Allelic Discrimination by Cq mode.
Allelic Discrimination by RFU mode.
CFX Manager™ software has two different display modes, quantification cycle (Cq) and relative fluorescence units (RFU). The Cq display mode displays the data as Cq values based on the selected thresholds; the RFU display mode displays the data in relative fluorescence units at the selected cycle.
Depending on your data set, the threshold may need to be adjusted. CFX Manager software automatically sets the threshold lines for discriminating alleles by taking an average Cq or RFU from your positive controls. If the run contains three controls in the plate, then the position of the threshold bars is based on the mean and standard deviation of the RFU or Cq of the controls. However, if the number of controls is less than three, then the position of the threshold bars is determined by the range of RFU or threshold cycle values in the selected fluorophore.
Some samples may be difficult to discriminate without positive controls. Ensuring that you have a positive control for each allele will help set the thresholds to discriminate between the different alleles.
Inaccurate calling of homozygotes as heterozygotes may be due to the causes below. Select one of the causes for possible solutions.
The ability to detect and amplify very small numbers of starting templates by PCR means that contamination control is always important. Contamination can cause reactions that should otherwise fail to be positive for the allele. Contamination in master mix components will show up in all samples. Contamination during plate loading typically affects one or a few wells.
The length of an amplicon can influence the amplification and melt profile especially 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).
Allelic discrimination requires well-optimized assays. Sometimes nonspecific amplification can occur, which may lead to false positive signals for one or more alleles. Nonspecific amplification can occur due to the loss of hot-start enzyme activity, degraded reaction components, or too much starting template.
The automatic calling of alleles is determined by the positioning of the threshold lines. It is therefore essential that these thresholds be set appropriately for the alleles to be called correctly. CFX Manager software typically positions the threshold automatically, but it may need to be adjusted in some cases. The autothresholds are most likely to be placed incorrectly when the variation among unknown samples differs from that among control samples. The Allelic Discrimination tab allows the data to be displayed as Cq values or RFU values, depending on your preference. Note that positive samples typically have lower Cq values and higher RFU values than negative samples.
It is important to use control samples with known genotypes. A variety of causes can lead to improperly identified control samples, including mislabeling, misidentifying the sample or the genotype, overgrowth or contamination of cultured material, and mutation.
Castellanos E et al. (2010). Rapid identification and differentiation of Mycobacterium avium subspecies paratuberculosis types by use of real-time PCR and high-resolution melt analysis of the MAP1506 locus. J Clin Microbiol 48, 1474–1477. PMID: 20129970
Gan XL et al. (2010). Association of an interleukin-16 gene polymorphism with the risk and pain phenotype of endometriosis. DNA Cell Biol 29, 663–667. PMID: 20662556
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