The Melt-Curve analysis are based on the GC content of the sequence. But, in case of 'T' to 'A' mutation or 'A' to 'T' mutation there is no change in GC content of the sequence. Then how can we identify 'T' to 'A' & 'A' to 'T' mutation using Melt-Curve analysis?
Have you checked? GC interactions just have a stronger bond and a bigger change in Tm, but HRM, although a little bit harder, should be able to pick up on A/T mutations as well.
It's likely as long as the design of assay is good enough. A/T mutation will not change the bonds but the position change (i.e., A-T swap the strand) would lead to small Tm difference of the amplicon for homologous genotypes. The heterozygous genotype will be easily detected as the melting curve can be quite different. You can read our paper published in 2008 to find out: High resolution melting analysis of almond SNPs derived from ESTs. Theoretical and Applied Genetics 118: 1-14.
Dear Yugendran
I agree, HRM is not based on the GC content alone and a T/A transition should be detectable using HRM profiling. If the difference is small, I recommend changing the settings of the HRM fluorescence calling intervals. The more frequent the calling during melting, the better resolution on differences in HRM profiling observed. This may extend your run-time depending on experimental design, but the desired result should be achieved. It would still, however, be crucial to run positive and negative controls with every run for comparison.
Good luck.
Nathaniel
I agree with Nathaniel about HRM profiling settings. In our experience like in A/T SNP allele specific Tm is lower than 0.2 celsius degree, we design primer to produce a shorter amplicon in order to increase possibility to separe allele specific melting curve. Sometime works sometime doesn't work
Dear Shu Biao Wu sir,
Can you kindly let me know the reason for difference in the Tm due to the A to T swap within the nucleotide strand. I went literature and could not find out the principle behind the change in Tm due to A-T swapping.
Dear Nathaniel Wade Mcgregor,
How does the fluorescence calling intervals affect the HRM analysis?
Looking forward to your reply.
Dear Cristian Araneda,
Thank you so much for your reply.
Is the minute change in Tm such as 0.2 celsius reproducable?
Dear Yugendran
Florescence is monitored at time-points during the high resolution melt - this is not a constant. The profile you get is therefore a representation of the collective florescence readings at particular monitoring points based on your HRM set-up. Small shifts in melt-profiles are harder to see when these reading points are spaced further apart. Taking readings more frequently during the melt will allow for better resolution/ distinction between amplicons with base-pair changes (e.g. your A/T transition).
I hope that clarifies things a bit better.
I haven't seen reports on firm reasons underlying the difference but it's possible that the difference of Tm is caused by the spacial differences of the molecules (A vs T) in the strand. This may lead to the slight difference of energy (heat here) required to break the bonds (A=T). This also depends how the neighbouring molecules are lined up. The difference can be undetectable sometimes depending on the conditions, i.e., amplicon sequence (GC content and secondary structure), length, PCR reagents, machine type, fluoresence dye, and melting profile etc. However, even if you can't resolve two homologous A/T genotypes straight away, you can manage to make artificial heterozygous to detect geneotype difference as homozygous genotypes is alway detectable. For example, by adding know AA genotype to the unknow samples you can make TT to AT. Once again, read our paper to see the resolution of ambiguous genotypes.
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