Mohsen,

I'm try to understand your problem, now I realize that there is no SNPs discovered in the exons you are working with.

I have performed SNP identification and genotyping in genes with no previous SNPs reported within them, but the methodology to be applied is different.

If you don't know if there is some SNPs, or if you don't where are them, you have to identify them first. There are various methods to identify thousands of SNPs (NGS, Arrays in some cases, databases), but if you are interested in just one gene or a few of them, I strongly recommend you to use direct sequencing of this exons. First, you have to design primers whose PCR product's length would be up to 1000 pb. The you have to sequence (conventionally) this amplicons in a small set of individuals (for example N =15), and then align the sequence to identify SNPs or potential ones. Then, for genotyping you have to design new set of primers, with an amplicon 70 - 120 pb in length, which flanks the SNPs identified. Now through HRM you will be able to genotype this SNP in your 2 experimental groups.

Agnieszka,

I have not certainty if your sample is degraded or maybe it is contaminated with another DNA. In both cases, HRM won't be performing well for this sample, so I recommend you to perform a new DNA extraction for it, or if you don't have the material to do that, just discard it (because you said is the only sample with this issue).

Regards!

I have some experience in HRM, and I never cleaned up the reaction from free dNTPs (they do not bind the dye used in the reaction). However, there are some limitations that can heve impact on overall results.

The DNA I am working with is a bit degradated and I have no possibility of making new DNA extraction. I thought it could be better to remove some short DNA fragments and dNTPs just for excluding the possibility I will have some non-specific artifacts.

The Rotor-Gene HRM template program and the Type-it HRM kit protocols both have the machine doing the pre-amplification and then immediately running the melt right after.

From what I can remember if you are running a concentration above 10 ng/microliter it suggest that you run 40 cycles, but if your amplification doesn't pick up until after 35 cycles to examine your results with suspicion (as that may be your short fragments and artifacts amplifying).

I have been running without purification and my results have been fine. I hope this helps!

I've tried HRMA with non-purified DNA (and a little bit degraded) at the Eco-Real time System (Illumina) and SteoOne Plus Mastercycler (Applied Biosystems), and it works very well.

Eva green is much better than SYBR green, SYTO9 is in second place for me..

I agree with Gustavo, but thought you might enjoy an explaination.

SYBR green is a non-saturating dye which means that that dye molecules can relocate and intercalate at the spaces in unmelted dsDNA regions causing a shift in the melting curve (in addition literature suggests that SYBR green can cause inhibition of the polymerases that you are using).

EvaGreen is a saturating dye that intercalates completely while the dsDNA is being built, which keeps released molecules from reincorporating.

I found this resource to be very useful while planning out my HRM assays:

http://www.europeanbiotechnologist.com/blog/wp-content/uploads/2010/12/HRM-tech-note.pdf

I am sorry, I am trying to understand your experimental design. Are you trying to make allele specific primer sets that will anneal to different SNPs?

In general for HRM you make a set of common set of primers with a SNP in the amplicon which is FLANKED by the primers. The amplification will be non-specific and the HRM will tell you the difference in the amplified region. The amplicon should be between 70-150 BP, with the closer to 70 the higher resolution you get.

When you run the HRM the melting curve will be shifted by between 0.2-0.5 degrees depending on the nucleotide substitution with a curve that is kind of a mix (will have a bump) for heterozygotes.

If you are looking to make an allele specific assay in which different primers will anneal to specific SNPs (usually the last base in the 3' end) I suggest looking into Tetra-ARMS PCR with AS-PCR as a guide to primer design. It is a much cheaper assay in which you only need to run a multiplex PCR and visualize your fragment size on a gel to determine which allele you have.

As far as annealing temps I generally go by the rule of thumb 4(#G+#C) + 2(#A+#T) = tm and subtract 15 for the annealing temp.

I'm running HRM on Illumina Eco: 40 cycles, HRM Kit from Bioline with EvaGreen. I have "correct" peaks (about 78C) with one additional peak above 80 degrees and third also above 80C. I checked amplicons on gel and I noticed that there are non-specific bands. Nonetheless they appear in one sample only - the oldest one, extracted with CTAB method. That's why I thought about DNA's degradation and non-specific annealing of primers.

Mohsen,

I'm try to understand your problem, now I realize that there is no SNPs discovered in the exons you are working with.

I have performed SNP identification and genotyping in genes with no previous SNPs reported within them, but the methodology to be applied is different.

If you don't know if there is some SNPs, or if you don't where are them, you have to identify them first. There are various methods to identify thousands of SNPs (NGS, Arrays in some cases, databases), but if you are interested in just one gene or a few of them, I strongly recommend you to use direct sequencing of this exons. First, you have to design primers whose PCR product's length would be up to 1000 pb. The you have to sequence (conventionally) this amplicons in a small set of individuals (for example N =15), and then align the sequence to identify SNPs or potential ones. Then, for genotyping you have to design new set of primers, with an amplicon 70 - 120 pb in length, which flanks the SNPs identified. Now through HRM you will be able to genotype this SNP in your 2 experimental groups.

Agnieszka,

I have not certainty if your sample is degraded or maybe it is contaminated with another DNA. In both cases, HRM won't be performing well for this sample, so I recommend you to perform a new DNA extraction for it, or if you don't have the material to do that, just discard it (because you said is the only sample with this issue).

Regards!

Thanks Gustavo. Yesterday I used DNA purifying Kit to clean samples. I'll check them today. Contamination is excluded, rest of SNP is working well...

Hi all

This is very interesting platform to share our thoughts, i am designing experiments to detect pathogens using real time HRM i do not wish to use any probe just dye only . My aim is to develop multiplex HRM for 5 pathogens so i am thinking to block 3 end of amplicon to create vairation in temperature melt among various amplicons. Anybody have use this concept? is there anything i should watch out for?

Thanks

cheers

I tried last week. Combination is hard and gives poorer results than separate analysis. On the other hand you spare the time so maybe it's worth of optimisation if you're going to do it long time with many samples. Otherwise I do not recommend it.