Oligonucleotides containing 2-aminoadenine and 5-methylcytosine are more effective as primers for PCR amplification than their nonmodified counterparts

Oligonucleotides containing the modified bases 5-methylcytosine and 2-aminoadenine in place of cytosine and adenine, respectively, have higher than normal affinity for complementary sequences. The strong binding oligonucleotides (SBO) are much better than their normal counterparts in PCR amplification: they yield significantly more product per cycle, allow amplification at annealing temperatures as high as 72°C and, unlike their normal counterparts, allow efficient priming from with a palindromic sequence. We propose that such strong binding oligonucleotides will be valuable in numerous PCR applications, including: (i) minimization of the frequency of mutants among PCR products; (ii) when only short specific primers can be designed based on available sequence information; (iii) when the material available for the analysis is limited in quantity; and (iv) when primer binding is blocked by DNA secondary structure involving a primer binding site, or chain extension is impeded by secondary structure in downstream sequences.