I have a problem with interpretation of PCR products’ length. The length of a template DNA sequence (human GSTP1 promotor) is about 1650bp and the length of a main PCR product is between 700-800bp. In silico PCRs show that the product should be specific. Ionic conditions (Mg), temperatures and time of PCR phases were changed in relatively wide range. Result always was the same.
However, when the right (reverse) primer is chosen to give a shorter product, about 1500, than the result is correct.
We also have used mfold to simulate if the template sequence is able to create some very stable structures. For average conditions 1.5mM Mg and 50mM Na at highest temperature of 95°C there can exist a strong stem-loop in the region of 1570-1590bp but it still doesn’t explain the product length in scope 700-800bp.
Do you have some opinions, suggestions useful to solve this problem?
Thanks for any help!