Increasing sample throughput on a standard 36-lane model 377 DNA sequencer to 48 or 64 samples per run.

During the last decade, automated DNA sequencing systems have started to gradually replace conventional methods of DNA sequencing and fragment analysis. Automated systems offer greater speed and ease of use, as they do not require a separate fragment detection step following electrophoresis. In addition, automated DNA sequencing and fragment analysis systems also offer substantial quality improvements over conventional manual systems: precision of fragment sizing, relative signal strength determination of different fragments, detection of mobility shift of fragments and data management are all improved. These benefits of automated DNA electrophoresis systems have led to ever-increasing demands on their usage. For the Model 377 DNA Sequencers (PE Biosystems, Foster City, CA, USA), optional upgrades of the basic 36-lane model to 48–64-lane or to 72–96-lane capacity can ease these pressures and increase throughput. However, upgrading a 36-lane Model 377 sequencer to a 48–64-lane instrument costs NZ $34800 (ca. US $18800), a sum many smaller institutions or laboratories cannot easily afford. Because the 48–64-lane upgrade involves only insubstantial changes to the detection electronics and control and analysis software, we reasoned that it might be possible, with minor alterations in work practice, to achieve 48–64-sample capacity using standard 36-lane hardware and software. This article describes our method, enabling the user of a 36-lane Model 377 sequencer to run 48 or 64 samples on a single gel without upgrading to a 48–64-lane model. Our laboratory uses both DNA sequencing and GENESCAN (PE Biosystems) fragment analysis, and we have found the method highly reliable and satisfactory for both applications. For sequencing reactions, samples were polymerase chain reaction (PCR)amplified from DNA isolated from fresh human blood, using primers specific to the HLA-A gene. PCR products were purified by Exonuclease I and Shrimp Alkaline Phosphatase (both from Amersham Pharmacia Biotech, Uppsala, Sweden) digestion at 37°C for 30 min (2). Purified PCR products were cycle-sequenced using BigDye (PE Biosystems) chemistry according to the manufacturer’s protocol (1). Sequencing reactions were ethanol-precipitated in PCR microplates, resuspended in formamide/loading dye and denatured at 95°C before gel loading (1). For GENESCAN fragment analysis, chromosome 10q23 microsatellite markers (PTEN/MMAC1 locus) were PCR-amplified from DNA derived from microdissected archival renal cell carcinomas using dye-labeled primers. PCR products were diluted between 1:2 and 1:16, combined with size-standard and deionized formamide/loading dye, denatured at 95°C for 5 min and quenched on ice before gel loading. Depending on whether a 48or a 64tooth comb was used, either 48 or 64 sequencing or GENESCAN samples were loaded in 3 or 4 batches, using an 8-channel multipipettor (SGE International, Ringwood, VIC, Australia). Samples were named 1–36 in the sample sheet. After run completion, lanes on the gel were tracked manually from 1–36 from the left side of the gel and extracted. Actual sample names were then entered in the sample manager Benchmarks