Abstract
Dye terminator cycle sequencing utilizes both unlabeled dNTPs and fluorescently labeled ddNTPS to PCR amplify DNA templates. In addition to labeling the DNA fragments, the modified ddNTPs also terminate the PCR extension, generating DNA fragments of varying sizes. Fragments are separated by capillary electrophoresis, during which laser excitation enables identification of each individual base. Dye terminator reaction kits are available from many scientific vendors. Here we compare Applied Biosystem™ (ABI) BigDye Terminator v3.1 cycle sequencing kit to Edge Biosystem™ BrilliantDye Terminator v3.1 cycle sequencing kit. A sample of BrilliantDye Terminator was received from Edge Biosystem for testing purposes. Both reagents were diluted, per our protocol, 1:128 or 1:10 depending on sample type. We sequenced 300 plasmid and PCR products in duplicate. Initial findings showed little to no difference between the two reagents for sequences shorter than 600bp. In sequences longer than 600bp, we noticed an increase in the length of high quality reads with the BrilliantDye Terminator compared to BigDye Terminator, achieving around 50-100 additional high quality, readable bases. We also saw that in some of the longer reads, there was a decrease in signal strength, but the raw data was more uniform and the traces were clearer when using BrilliantDye. Overall, in our opinion, the BrilliantDye Terminator and BigDye Terminator performed similarly for sequence reads up to 600bp, but the BrilliantDye outperformed BigDye in sequences greater than 600bp.