PCR problems - (Jun/23/2002 )
I have two very similar sequences and have to pick up oligos for PCR, which sould be able two amplify one seq. and not to amplify another, but all oligos, looking rather distiguishly (especially on 3`termini) in two sequences in OLIGO amplify both seq-s presenting the same size amplicons. Than I found restriction endonuclease HindIII, which was able to cut my DNA in the middle of amplicon of one seq, and didn`t cut another, and did PCR, but still got the same result of amplification both. May be the sticky termini could do self- ligation? Anyway any ideas will be appreciated.
No restriction digest will be 100% complete. There will always be remaining uncut template to contaminate your PCR. Depending on the purity of your oligos there may be left over impurities from the oligo synthesis that lack your 3' difference. You may want to have the oligos HPLC purified before this sensitive application.