It is because of too much DNA input to PCR, and amplification is seen from 1st cycle. Reduce number of steps in first PCR (10-15?). Usually nested PCR is done with different sets of primers to increase specificity. Is this an intercalating dye assay? Then, be cautious on NTCs.

Many thanks,

I have designed different sets of primary and nested primers for the nest HRM assay. I would also reduce the number of cycles in the 1st PCR (30 is okay?). 

You can adjust the number of first PCR cycles based on target concentration in original sample. Find this number, such that you get Ct value approx 20-25 in your second PCR (just a guideline only, no hard rules here)

hi Kim,

What was the dilution factor that you used for the second pcr? When I did some nested snp assay taqman rt pcr I used a 1000 fold dilution of the first pcr as input fraction for the second pcr.

cheers

Hi Olivier,

In the second PCR, I put 1μL non-diluted template taken from the 1st PCR. I also think about the 1st PCR product dilution as you said. I also measured the 1st PCR product concentration and it ranged around 0.35μg/mL. Do you have any idea about the dilution level , 1000 fold?

Best regards,

Hi Mr. Manoj,

Today I run 1st PCR 20 cycles and then take 1μL non-diluted template for the 2nd HRM. The amplification is good, only 2 case show poor amplification compared with the others. On HRM analysis, cases showing good amplification has HRM result the same as result of direct sequencing (I chose WT control as reference and also put mutant control to compare). However, 2 cases showing poor amplification above showed different HRM result compared with direct sequencing. 

Anyone has any solutions for this?

Manythanks

Good. Can you post the HRM curves? Also, mark the samples that failed, on above plot.

Here they are. I ran 4 cases which had Sanger sequence result to compare. Well design and protocol also included. Primers NRAS codon 61 designed by primer blast NCBI (124bp).

Hi Olivier,

I laso tried serial 1000 fold dilution and run the second HRM. The amplification is better (please check image). But the HRM result of all 4 cases are the same as the previous run. 2 cases CRH01 and 03 showed mutated result in both 2 HRM . However, Sanger sequence result is WT for these 2 cases. CRH02 and 04 have the concordant results between Sanger and HRM. So I want to check for Sanger sequence again because Sanger does not have good sensitivity compared with other methods.

Hello, 

I just had a quick question about this subject. Do you do a PCR purification step after the first PCR or is this not necessary? 

Hello.

No, I did not perform PCR purification after the first PCR. However, due to the heterogenity and poor quality of the DNA extracted the FFPE samples HRM analysis showed abberant and unreliable curves though I tried to normalize the nested template input.