| Description of problem |
Likely cause(s) and suggested solutions |
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Insufficient Amplification
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PCR cycle number
If, after the recommended cycling protocol, the signature genotyping groups have not yet formed (i.e. amplification is incomplete), it is advisable to thermo-cycle the samples further and re-read on the fluorescent plate reader. Three further PCR cycles should be used (of the type of the last 26 cycles in section 7 i.e. 94°C for 20 seconds and 55°C for 60 seconds) followed by re-reading on the plate reader. This re-cycling process can be repeated until the desired grouping is achieved. The re-thermo-cycling / re-reading process can be carried out on a plate that has been stored at room temperature for as long as one week.
G/C percentage
Low G/C (G/C <30%)
Where the G/C percentage of the assay oligos is low (G/C <30%) slow amplification can result. Increasing the final MgCl2 concentration in the reaction mix from 1.8mM to 2.2mM or even 2.5mM can compensate for this.
High G/C (G/C >70%)
Assay oligos with a high G/C percentage (G/C >70%) can also be problematic. Where this situation is encountered, running the assay at the standard MgCl2 concentration (1.8mM) with the addition of 5-10% DMSO to the final volume of the assay will usually provide a solution. When adding DMSO to the reaction, there is no need for a concomitant reduction in water volume to compensate for the disturbed reaction volume.
Low DNA concentration
DNA concentration may be lower than expected causing samples to take longer to amplify. This can sometimes be addressed by simply cycling the samples further (see section above), though too many extra cycles may result in amplification of the negative control samples. It is preferable to repeat the genotyping with DNA in the recommended concentration range (1-40ng/µl). It is recommended that the Picogreen dsDNA fluorescent quantification system be used in preference to spectrophotometric methods, as the latter tends to overestimate DNA concentration in this range. Alternatively, a more pragmatic approach can be used: representative test samples can be genotyped at a variety of dilutions and the optimal dilution selected.
High DNA concentration
Conversely, samples of very high DNA concentration can also cause poor / no amplification. Where this is the case, it is because there is also a high concentration of PCR-inhibiting contaminants present. The solution to this is simply to dilute the samples such that the contaminants are diluted to non-inhibitory levels.
DNA samples dissolved in a buffer that contains EDTA
After purification, DNA samples are often eluted / diluted in TE buffer, which contains EDTA. EDTA chelates Mg2+ ions leading to insufficient Mg2+ for the reaction to proceed. However this can be overcome by increasing the MgCl2 to compensate. For example, if the DNA samples contain 1mM EDTA, in a 4µl reaction, where the DNA samples account for 50% of the reaction volume, addition of extra Mg2+ to the amount of 0.5mM will resolve the issue. A greater problem is when the DNA samples have been diluted such that they contain non-uniform concentrations of EDTA. In such a case, EDTA should be added to the NTC wells.
Assay mix storage conditions
Assays that have been subjected to multiple freeze/thaw cycles or otherwise stored incorrectly can lead to poor amplification in the subsequent reaction.
It is advisable to divide the mix into aliquots to avoid multiple freeze/thaw cycles. Incorrect storage, or multiple free/thaw cycles will negatively affect the performance of the assay.
Aliquots of assay can be safely stored at 4°C for 1-2 weeks, approximately 1 year at -20°C or indefinitely at -80°C.
Reaction Mix storage conditions
As with assay mix storage, it is essential that the Reaction Mix is stored as directed.
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Amplification Too Fast
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High DNA concentration
If the reaction is proceeding too quickly, it may be an indication of too much DNA in the reaction, causing samples to amplify more quickly than expected. To resolve this, dilute the DNA samples to the recommended range of 1-40ng/µl (as determined by Picogreen).
Magnesium concentration
Alternatively, it may be that there is too much magnesium in the reaction for the G/C percentage of the assay being genotyped. Inclusion of 5 -10% of DMSO will generally rectify this. Alternatively reduce the amount of DNA in the reaction or reduce the number of PCR cycles in the second phase from 26 to 20 (see the KASP genotyping manual), such that the total number of cycles becomes 30. |
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Scattered grouping of genotyping calls
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Scattering of the genotyping groups can be caused by a variety of factors:
Cross-contamination between the DNA samples on the plate can cause genotypes to cluster outside the expected genotyping groups.
Too much magnesium for the SNP being assayed is another factor that can lead to scattered grouping. A reductive magnesium titration may resolve the issue.
Poor DNA quality (and inconsistent quality across the plate) can lead to this effect.
TempliPhi WGA DNA used as the template DNA. In some cases scattered heterozygous groups can be seen with WGA amplified DNA. Re-WGA with a DOP based method may well rectify this, such as the KlearAmp product available from KBioscience.
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Little / no separation of the hetero- and a homozygote group
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An imbalance in the allele-specific primers can result in the heterozygous group migrating towards one of the homozygote groups, making genotype scoring difficult.
Such an imbalance can occur for a variety of reasons: poor synthesis of one of the allele-specific primers, accidental addition of an insufficient quantity of the allele-specific primers when making the assay, or more efficient amplification of one allele-specific primer compared to the other. Regardless of cause, the problem can be ameliorated with an increase in the concentration of the alternate allele-specific primer. In the example shown on the left, allele-specific primer 1 leads to the blue group, allele-specific primer 2 giving the red group. In increase of around 40% of the AL-spec 1 primer would cause the green heterozygous group to move to a more central location on the plot, allowing more confident scoring of the genotypes.
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Heterozygous group migrating to a much lower position (with respect to the homozygous groups) on the x- or y-axis than expected
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The cluster plot shows an example of the effect that occurs when the assay aliquots are thawed without subsequent mixing (or not mixed before they are aliquoted) causing the forward primers to saturate the fluorescent quenching system. The effect is characterised by the genotyping clusters seemingly adopting incorrect positions on the plot (despite often clustering well into groups).
Whilst mixing the assay aliquots after thawing and before use is strongly recommended for the reasons described, it is worth noting that the problem could also have occurred when assembling the assay if the constituent primers were hydrated and then frozen /thawed without proper mixing.
The solution to the problem is to remake the assay with sufficient mixing of oligonucleotides
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Heterozygous cluster too close to the origin
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A related problem to that described above is encountered when the homozygous cluster positions appear to amplify correctly but the heterozygous group amplifies less than might be expected, remaining close to the origin. This is also caused by the forward primers saturating the quenching system and can be resolved by remaking the assay with a reduced concentration of both forward primers (instead of 12µM, try 8µM).
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Too many genotyping groups
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Multiple genotyping clusters caused by the presence of polymorphism(s) within primer binding site(s). In diploid organisms, more genotyping clusters can be observed when primers (forward or reverse) are designed such that they bind to a region containing other polymorphism(s). Such non-target polymorphism causes differential PCR amplification efficiencies, making confident allocation of the genotypes less reliable. In the example shown, the reverse primer was designed in a region containing another polymorphism. The issue can be resolved by re-locating the primer(s) to a region containing no polymorphisms, if one is available. Alternatively, a wobble base can be inserted in the primer sequence at the site of the neighbouring (non-assayed) SNP. Duplicated diploid or polyploid genomes However, when assaying duplicated-diploid plant genomes, up to five genotyping clusters are potentially possible and does not mean that the system is misreporting. |
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Fewer genotyping groups than expected
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Some assays will report just one genotyping cluster (monomorphic genotyping), which can be a genuine result if the population being analysed contains only one genotype with respect to the (probably low frequency) SNP being studied. However, monomorphic data can also occur because the SNP is not real. Another reason for one group appearing can be that the primers are hybridising to a homologous region in the genome. Redesigning the assay to the opposite strand can often resolve this. |
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Some samples not amplifying at all
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Inconsistent DNA quality / quantity If some of the samples on the plate are of a much lower concentration and / or purity that the other samples, the observed effect may be seen due to some of the samples amplifying and some not. This is more likely if the DNA samples being analysed on the same plate are from different sources. Arraying / dispensing This pattern can be seen if the arraying of the DNA into the PCR plates was done carried out correctly (such that there is little / no DNA in some wells supposed to contain it. Alternatively, poor dispensing of reaction mix into the wells could lead to the same effect. This possibility can be checked by viewing the ROX levels in each of the wells across the plate as this relates directly to the volume of the assay / reaction mix combination that was dispensed into the plate. |
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Misreading of data
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Even if the KASP genotyping reaction has proceeded correctly, the resultant data can still appear incorrect. Possible causes are: Plate reader fault The plate reader may have developed a fault Incorrect plate positioning Check that the plate is inserted correctly into the plate reader (or if not correctly placed on a plate holder). Temperature of plate reading The plate reader could be reading at an elevated temperature. Because of the mechanism of action of KASP (see the KASP genotyping manual), KASP data must be read at 40degC or below (ambient temp is preferable). Reading data at temperatures higher than this will dissociate the quencher-bound oligo from any remaining unincorporated fluor-bound oligo and cause a meaningless signal. |
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Genotyping groups too close together / merging
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Specificity I Genotyping groups close together indicate that the specificity of the KASP reaction is not sufficient. In this instance, the two allele specific primers are, to some extent, able to bind to each others' sites and hence the genotyping cluster positions are closer to each other than they should be. Experimentally increasing in the annealing / extension temperature in the thermo-cycling program will solve this problem as it will force improved specificity. |
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One genotyping group in the top right hand area of the plot
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Specificity II Where only one the observed genotyping group appears, the problem is related to specificity. In the case shown in the diagram, the assay is completely non-specific, i.e. the allele-specific primers are not able to bind specifically, causing a completely mixed fluorescent signal. This situation requires a re-design of the assay. For complicated homologous genomic sequence please contact KBioscience as we are often able (with some work) to produce a working assay. |
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Skewed positioning of genotyping groups
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Homology The skewing effect is caused by binding of the KASP primers to a sequence homologous to the one intended for analysis. If the KASP primers are able to bind elsewhere in the genome, amplification will occur there as well. However, the SNP may well not exist at the alternate binding site(s) so the eventual signal output of the assay will be influenced, causing the skewed grouping effect. KASP primers binding in identical regions The problem occurs because the region being assayed is not unique in the genome. However, small sequence differences between the homologous sites often exist. The solution therefore is to redesign the assay taking advantage of any sequence differences between the desired region and the others, such that the 3' end of the common (reverse primer) is unique to the region being analysed. KASP primers binding in similar regions The KASP primers may bind in very similar regions in the genome (rather than identical regions) in which case the issue is specificity and it is possible that the situation could also be resolved by simply increasing the annealing / extension temperature. Alternatively, a two-step solution can be used whereby an initial PCR is carried out to specifically amplify the region of interest, prior to genotyping. Providing the primers chosen for the initial PCR are known to uniquely amplify on the region of interest, the resultant amplicon from the initial PCR can then be used as the template DNA. A variety of experimental dilutions of the amplicon DNA may be required to find the best concentration to use. |
