Cost savings using automated DNA sequencing.

Confirmation of mutations mostly involves DNA sequencing. The conventional manual radioactive labeling sequencing protocol may be conceived as the method of choice because of the perception that the capital and operational costs of automated sequencers are high. Velickovic et al. (3) reduced the cost of DNA sequencing by running 48 or 64 samples on a 36-lane model 377-01 ABI PRISM automated sequencer (PE Biosystems, Foster City, CA, USA). We found that a cheaper model, the 18-lane model 377-18 ABI PRISM, can run 37 samples on one gel. We also found that the volume of the most costly reagent, dye terminator reaction mixture, can be reduced to cut operating costs by 69%. As a result, automated sequencing is more feasible for use in research or clinical service. At least 37 samples can be loaded on a 377-18 sequencer by using lanes 15–51 of an aligned 64-well shark’stooth comb. Since the wells are narrower than for an 18-well comb, the amount of sample required to get the same intensity of signal is reduced. Purified PCR products were cycle sequenced using the dRhodamine Dye Terminator FS Cycle Sequencing Ready Reaction Kit (PE Biosystems) in a total volume of 5 μL instead of the suggested 20 μL. We detected a heterozygous mutation in either a 1.5-μL aliquot from a 20-μL reaction loaded on an 18-well comb or a 0.7-μL aliquot from a 5-μL reaction loaded on a 64well comb (data not shown). The signal quality remained the same. By our protocol, we screened over 2000 PCR samples for novel mutations in the TIGR (2) and PAX6 (1) genes. Both capital and reagent costs are reduced. A 36-lane 377-01 sequencer costs about $184000 US, while a 37718 costs about $100 000 US, a capital cost reduction of 46%. The PE Biosystems protocol costs $6.96 US per sample ($6.36 for dRhodamine dye terminator, $0.40 for ExoI and SAP and $0.20 for acrylamide and other consumables), while our protocol costs $2.19 US ($1.59 for dye terminator and $0.60 for the rest), a reagent cost savings of 69%. Automated sequencing has advantages over manual methods, in addition to those mentioned by Velickovic et al. (3). It avoids the higher cost and environmental hazards of radioactive labeling. It can read longer sequences and requires fewer reactions to cover the same length. Our modifications increase the capacity of the machine and reduce operating costs, allowing laboratories with limited budgets to more readily achieve their research targets.