SUBSTRATE NUCLEOTIDE-DETERMINED NONTEMPLATED ADDITION OF ADENINE BY TAQ DNA-POLYMERASE - IMPLICATIONS FOR PCR-BASED GENOTYPING AND CLONING

The Applied Biosystems PRISM(TM) florescence-based genotyping system a s well as the Invitrogen TA Cloning(R) vector system are influenced by the tendency of Taq DNA polymerase to add an adenine nucleotide to th e 3' end of PCR products after extension. Incomplete addition of adeni ne to a majority of PCR product strands creates problems in allele-cal ling during genotyping and potentially diminishes the cloning efficien cy of such products. Experiments reported here show that certain termi nal nucleotides can either inhibit or enhance adenine addition by Taq and that PCR primer design can be used to modulate this activity. The methods we propose can substantially improve allele-calling for proble matic microsatellite markers when using GENOTYPER(TM) software.