Any scientist that has used the traditional Sanger DNA sequencing method is aware that the process is tedious, time consuming and messy due to the involvement of radioisotopes and harsh chemicals. The ABI 3730xl DNA Analyzer offers an alternative in the form of a fully automated system. The protocol involves a PCR reaction using a very robust Mastermix (BigDye from Applied Biosystems), reaction purification and plate loading on the 3730xl. The instrument does all subsequent analysis, requiring only operator loading of 96-well plates.
Several advantages were noted for the ABI 3730xl system. First, the accompanying software package is intuitive and comprehensive. Also, up to sixteen 96-well plates can be loaded on the stacker for high-throughput automated processing. Although attention is required to ensure adequate buffer, polymer and waste levels are maintained, this instrument was extremely hands-free. The actual operation of the instrument, even data analysis, was very simple. The only precursory step to the sequencing reaction is a spectral calibration that ensures all capillaries are functioning properly and the signal is strong enough for detection.
The 3730xl uses the BigDye Terminator reagent that is the most stable and most consistent in the market today. This chemistry is also the most prevalent, which has lead to many laboratories to tinker with it so that there is a wealth of knowledge about what can and cannot be done with it. Thanks to the extensive use of the BigDye Terminator reagent by many core labs, scientists have been able to dilute this dye significantly. Although this needs to be validated on a per-lab case, the savings that can be made are very significant.
While reagents like the BigDye Terminator can be relatively cheap when diluted, there are other components of the instrument that are expensive. A 96-capillary array can be used for about one hundred runs or so and it can be pushed for more with concomitant decrease in sequence read quality. Each one of these capillaries is not cheap. In addition, the polymer that is used to run the instrument gets used up very fast, and that too is not cheap.
The footprint of the 3730xl is only 100 cm x 73 cm x 89 cm (WxDxH). While not small, it occupies a relatively small area compared to the amount of data it generates. It can be placed on a table top or the side of a bench just like you would an average sized table-top centrifuge (at 180 kilograms (almost 400 pounds), the 3730xl is not a machine that you want to move around). Also, the 3730xl generates a considerable amount of heat, so it is advisable to install vents and keep the air conditioning running, even during the times when the machines is running multiples plates through the night. Failure to do so can adversely affect the quality of the sequencing reads. The instrument needed to be cleaned regularly to sustain top performance, thus maintenance can be time consuming. Maintaining the system required that the polymer blocks were removed and rinsed with water, frequently posing issues when connections were not properly reassembled. In addition, the polymer was not very stable at room temperature when the instrument was not used at full capacity, thus requiring replacement. For this reason, even short breaks in instrument usage required a water rinse to clean residual polymer. All of the maintenance steps were more labor intensive than intellectually challenging, since the software walks you through the process step by step and the user manual was a great resource, as well.
The ABI 3730xl DNA Analyzer remains the workhorse of our lab for high throughput DNA sequencing. With a capacity of about 1 million base pairs per day, it not only provides an incredible capacity, but also flexibility.
Ivan Delgado, Ph.D.