I am having some problems at an AS-PCR of the TNFRSF1A gene. I have two samples from a couple, which I have already sequenced the region of interest: one is heterozygous for the variant (mother) and the other is WT (father). I want to perform an AS-PCR in separate tubes (one with the WT and the other with the MT), but the MT primer pair is amplifying in both samples.
Initially, I designed a WT-forward and a MT-forward primers with a mismatch at the third base from 3’end and the MT-forward 3’end base complementary to the variant, according to Liu (2012), as well as a Common-reverse primer. However, as the MT primers seemed to be nonspecific and amplified both the parents, I redesigned a WT-forward and a MT-forward primers with a mismatch in the second base from 3’end and the MT 3’end base complementary to the variant, using WASP.
The fragment is ~1200bps, so I initially used a LongRange Enzyme, until I knew that it has proofreading activity and it might be affecting my results. I tried to use TaKaRa using the methodology from Pont-Kingdon (2004), although my fragment is considerably smaller.
I keep having nonspecific amplification of the MT fragment of the father.
Any suggestions?
Thanks!
PS.: I detected a de novo duplication in their son through qPCR (he is also heterozygous for the variant used in AS). I tried to sequence the fragment harboring the duplication, but I'm having difficulties. I was trying to amplify each allele in separate to sequence and characterize the duplication, but since I have few sample from the kid I am trying to first optimize the reaction using samples from the parents.
design a reverse primer much nearer to the as primer .Use a normal cheap taq polymerase and run a temperature gradient of the annealing temperature and there should be temperatures at which each primer only amplifies the correct sequence and the other sequence does not amplify
Hello Ines,
I agree with Paul. With WASP you are much more likely to get non-specific fragments the longer the target fragment. In the past I have always targeted
Thank you for your suggestions! However, I really need to amplify a big fragment (the minimum I can amplify is ~800bps), since I'm trying to amplify each allele in separate wells to sequenced them separately. Should I try a PCR with a HotStar Taq Polimerase and a gradient starting with high annealing temperatures?
Why are you trying to sequence them separately?
Gradient starting with high annealing temperatures is a good place to start.
I detected a de novo duplication in their son through qPCR (he is also heterozygous for the variant used in AS). I tried to sequence the fragment harboring the duplication, but I'm having difficulties. I was trying to amplify each allele in separate to sequence and characterize the duplication, but since I have few sample from the kid I am trying to first optimize the reaction using samples from the parents.
Hi, I think the best way to do this is by performing a high-resolution melting analysis. With this method, you can find variations as small as from one single nucleotide.
Find more info at:
http://www.lifetechnologies.com/es/en/home/life-science/pcr/real-time-pcr/real-time-pcr-applications/genetic-variation-analysis-using-real-time/high-resolution-melting-hrm.html
Good luck with your research,
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