Dear colleague
Does the primer can produce PCR product if Single Nucleotide Polymorphism was located in primer sequence?
if you look up DCAPS it is a method ( the site designs your primers) where almost any snp can be turned into a restriction site by the deliberate addition of a second base change built into the primer. Better than 99% of changes can be turned into a RE site and the cutting of that site confirms the presence of exactly the natural base change that you are investigating
If your change already is a RE site then just design primers 2/3 and 1/3 of the amplimer about that site so a 300 base fragment will cut to 100 and 200 bases which will separate well on all agarose concentrations. The sizes I chose are just approximations...any 2 fragments that separate well will do
Thank you for your recomendation. Therefore, how about restriction site in primer? Can the primer produce PCR product if nucleotide mutation occured in the primer?
if you design a restriction site in the primer it will incorporate into the amplimer and will cut ok if you add 3 extra bases at the end of the primer.Enzymes cut badly if the cut site is exactly the end of the primer. You cannot include the RE site when calculating the annealing temperature of the primer only the part of the primer that binds to the original dna sequence. Mutations only occur in the primer if you design it to be there
OK. Thanks for your information. I will send you a journal that has restriction site in primer, but the PCR and RFLP products of IGF1/SnabI gene were cleared. I need your suggestion about using the primer based on this journal or make new design primer. Thank you
The reverse primer in this paper is based on the dcaps method. 4 bases in from the 3' end there is a deliberate mismatched base ( an A). When this primer works then it will create an SnabI site TACGTA only when the afo17143 base is a T. then when you cut with the enzyme you will get 220 and 246 if there is a heterozygote TC only 246 if there is homozygous C and only 220 for homozygous T.
Use their Tm of 62 c. I would use shorter denaturation times 30secs and annealing times 15-30secs but their method looks good and it works so there is no need to change anything really. Always run a positive and negative control using this type of technique to check both the enzyme activity and the possibility of pcr contamination
Thank you very much. I am very happy for your response. I will contact you again if any problem in PCR RFLP work. Thank you very much
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