Capillary Failure

Capillaries fail and need to be replaced after a number of sample injections. One of the primary reasons for capillary failure is that the dynamic coating on the inside wall of the capillary fails to work properly. Capillary failure is diagnosed by the presence of abnormally broad peaks that define a loss in resolution between closely spaced STR alleles. This loss in resolution is most likely the result of DNA and enzymes from the injected samples adhering to the capillary wall (Isenberg et al. 1998).

Capillaries can be removed from the ABI 310 and regenerated with consecutive washes of water, tetrahydrofuran, hydrochloric acid, and polymer solution (Madabhushi 1998). Margaret Kline at the National Institute of Standards and Technology (Gaithersburg, MD) has developed a capillary regeneration procedure that involves forcing several milliliters of deionized water and then a Tris-EDTA buffer through the capillary to remove any material that has bound to the inner wall. Of course the capillaries have to be removed from the instrument in order to perform this procedure. However, capillary lifetimes of over 500 injections have been repeatedly demonstrated when using these wash steps. Some labs though may find it more convenient to just replace the capillary at around 100 injections per the manufacturer's suggestion. Unfortunately, the ABI 310 does not permit an on-the-instrument wash that could be used to recondition a capillary and eliminate the need for frequent capillary replacement.

STEPS PERFORMED BY THE STANDARD MODULE

Instrument operation and data collection on the ABI 310 Genetic Analyzer is controlled by a series of steps and procedures that grouped together are referred to as a 'module'. The standard module used for STR typing is titled 'GS STR POP4 (1 mL) F'. The steps for this module are listed below with an explanation for the purpose of each procedure.

Prior to starting the regular cycle of filling the capillary with polymer solution and injecting and separating DNA samples, several steps are performed in the standard module. First, the temperature on the capillary heating plate is brought up to 60°C to thermally equilibrate the capillary. The laser is turned on to full power (~10mW). The autosampler platform is moved around in order to verify that the instrument is working well. The following steps are then performed with each sample that is analyzed on the ABI 310 capillary system:

■ Capillary fill - polymer solution is forced into the capillary by applying a force to the syringe; the syringe position moves down by 5-10 revolutions or steps per injection with POP-4 and a 47 cm capillary. If the syringe moves significantly more than 10 steps, then there is likely a leak in the pump block; if less, then the capillary may be plugged.

■ Pre-electrophoresis - the separation voltage is raised to 10 000 V and run for five minutes. This step helps check for bubbles inside the capillary and helps to equilibrate the system for sample separation. If there are bubbles inside the capillary, the current will remain at zero when the voltage is raised because ions are not flowing through the capillary.

■ Water wash of capillary - capillary is dipped several times in deionized water to remove buffer salts that would interfere with the injection process.

■ Sample injection - the autosampler moves to position A1 (or the next sample in the sample set) and is moved up onto the capillary to perform the injection. A voltage is applied to the sample and a few nanoliters of sample are pulled onto the end of the capillary. The default injection is 15 kV for five seconds.

■ Water wash of capillary - capillary is dipped several times in waste water to remove any contaminating solution adhering to the outside of the capillary.

■ Water dip - capillary is dipped in clean water (position 2) several times.

■ Electrophoresis - autosampler moves to inlet buffer vial (position 1) and separation voltage is applied across the capillary. The injected DNA molecules begin separating through the POP-4 polymer solution.

■ Detection - data collection begins. Raw data files are collected with no spectral decon-volution of the different dye colors. The matrix is applied during Genescan analysis.

This entire process is accomplished in approximately 30 minutes per sample from one injection to the next assuming that the default time of 24 minutes for electrophoresis is used. The overall time for the capillary fill and pre-electrophoresis steps is about six minutes. DNA fragments up to approximately 400 bp in size should be through the capillary within 24 minutes of electrophoresis at 15 000 V on a 47 cm capillary (320 V/cm).

ALTERNATIVE SOLUTIONS FOR HIGHER THROUGHPUT CAPABILITIES

Each ABI 310 instrument is capable of routinely analyzing about 8000-10 000 sample injections per year. For laboratories desiring to process higher volumes of samples, multiple ABI 310 instruments or alternate analysis platforms may be used. Alternative electrophoresis instrumentation platforms from Applied Biosystems with the same multi-color detection technology include the ABI 377 slab gel system, the 16-capillary ABI 3100, the four-capillary ABI 3100-Avant, and the 96-capillary ABI 3700 and 3730.

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