Nucleotide analogs facilitate base conversion with 3 ' mismatch primers

We compared the efficiency of PCR amplification using primers containing ei ther a nucleotide analog or a mismatch at the 3' base. To determine the dis tribution of bases inserted opposite eight different analogs, 3' analog pri mers were used to amplify four different templates. The products from the r eactions with the highest amplification efficiency were sequenced. Analogs allowing efficient amplification followed by insertion of a new base at tha t position are herein termed 'convertides', The three convertides with the highest amplification efficiency were used to convert sequences containing C, T, G and A bases into products containing the respective three remaining bases. Nine templates were used to generate conversion products, as well a s non-conversion control products with no base change. We compared the abil ity of natural bases to convert specific sites with and without a preconver sion step using nucleotide analog primers. Conversion products were identif ied by a ligation detection reaction using primers specific for the convert ed sequence. We found that conversions resulting in transitions were easier to accomplish than transversions and that sequence context influences conv ersion. Specifically, primer slippage appears to be an important mechanism for producing artifacts via polymerase extension of a 3' base or analog tra nsiently base paired to neighboring bases of the template. Nucleotide analo gs could often reduce conversion artifacts and increase the yield of the ex pected product. While new analogs are needed to reliably achieve transversi ons, the current set have proven effective for creating transition conversi ons.