the results of allele specific PCR is the other way around - I dont know why (Dec/07/2006 )
Hello,all. Currently, I am doing allele specific PCR to amplify DNA from only one allele. The DNA template is from homozygous mouse and the expected results should be one band only in the reaction which the primer match the DNA template. But the results is the other way around: There is a strong band from the reaction in which primer and template did not match and the band from the reaction in which template and primer match is weak and unspecific.
I am sure I did not make mistake during experiment. The allele specific primer is the reverse one and there are two base different (AC in one and CA in another) at the 3'tail. they share the same forward primer. The length of DNA template is 230 bp. The melting tempature for forward primer is 61.3 C and the melting tempature of the two allele specific reverse primers is 49 C. I used 38 cycles. The annealing temp I have tried is 56, 60, 61, 62. But the results are quite similar. weak and unspecific bands in the reaction containing correct primer and template and strong but unspecific bands from wrong primer and template.
Does anyone have any suggestions? I hope it is not the mistake of sequencing.
If you are absolutely confident in your primers etc. I suggest doing a PCR with primers that stop short of the allele. Amplify it, clone it and seq it. The you'll know if the template is right, seq etc. I have done allele-specific PCR with one base different and it worked fine.