PEAK HEIGHT VARIATIONS IN AUTOMATED SEQUENCING OF PCR PRODUCTS USING TAQ DYE-TERMINATOR CHEMISTRY

Direct sequencing of PCR products using Tag DNA polymerase with dye-la beled dideoxy chain terminators results in traces with uneven peaks. T he peak height variations reflect the disproportionate rate of incorpo ration of deoxynucleotides vs. their analogs, a phenomenon that is hig hly dependent on the neighboring DNA sequence. Such peak height variat ions make it difficult to call bases correctly or to interpret whether or nor a polymorphism is present. We have systematically examined pai rs of sequence-tagged sites that vary at only one nucleotide to determ ine how a single base change will affect the peak heights of neighbori ng bases. We have found that the peak height of a particular base can often be predicted from the knowledge of just one or two nucleotides 5 '- to the base in question. We have also observed several artifacts th at occur consistently in the sequencing traces. These artifacts can be misinterpreted as polymorphisms or can obscure the real peak at that site. The empirically derived trends presented in this report can be u tilized profitably when one is editing sequence data or examining them for polymorphisms and mutations.