Capillary

Buffer

(inlet)

Autosampler Tray

Deionized water

Figure 14.1

Schematic of ABI Prism 310 Genetic Analyzer. The capillary stretches between the pump block and the injection electrode. The mechanical stepper motor pushes polymer solution in the syringe into the pump block where it enters and then fills the capillary. Samples placed in the autosampler tray are sequentially injected onto the capillary. Electrophoretic separation occurs after each end of the capillary is placed in the inlet and outlet buffer and a voltage is applied across the capillary. A laser (not shown) is used to detect fluorescently labeled DNA fragments as they pass by the capillary detection window.

3. Filling the buffer reservoirs so that current can flow between the electrodes and separations occur across the capillary; and

4. Placing deionized water in tubes that will be used for keeping the end of the capillary wet and cleaning off buffer salts before and after the injection process.

Each of these components plays an important role in the CE process and will be described in greater detail below.

THE CAPILLARY

At the core of a CE system is the capillary, which is a narrow glass tube that has an inner diameter of 50, 75, or 100 |lm. Capillaries typically used for STR separations on the ABI 310 have an inner diameter of 50 |m. The outside of the capillary is usually coated with a plastic polyimide jacket to allow users to handle the capillary without it breaking. However, this coating is opaque and thus inhibits optical detection of anything passing through the interior of the capillary. A small capillary window is therefore needed to observe the separation products and is usually generated by burning away several millimeters of the polyimide coating. Capillary with windows already created in them may be purchased or a user can buy a long roll of capillary and burn their own detection windows. Once the fused silica (glass) tube is exposed the window region must be handled carefully as it is easily breakable.

Two factors that are impacted by capillary length include peak resolution and separation time. Generally, the longer the capillary the better the resolution, but the greater the separation time. With CE, two capillary length measurements are important. The length-to-detector (Ldet) is a measure of the distance from the capillary inlet where the DNA sample is injected to the detection window where the laser shines on the capillary and the fluorescent dyes are excited and detected. The total capillary length (Ltot) is the complete length of the capillary or in other words the distance from inlet to outlet buffer solutions when electrophoresis is occurring.

Varying the Ltot distance impacts the electric field strength that is applied to the CE system while shortening the Ldet improves the separation speed. For optimal performance (i.e., highest resolution) with CE, it is best to have Ldet as close to Ltot as possible. However, instrument space constraints keep Ldet shorter than Ltot usually by 7 cm or more, primarily because the electrophoresis electrodes cannot be too close to the detection electronics. The detection window on the ABI 310 is fixed 11 cm from the outlet buffer. There is a minimum capillary length (Ltot) of approximately 41cm (i.e., 30 cm Ldet) with the ABI 310 Genetic Analyzer.

Applied Biosystems has made it easy for the user by supplying two lengths of capillary: 47 cm (36 cm to detector) and 61 cm (50 cm to detector). The shorter capillary is typically used for GeneScan applications, such as STR typing, where a faster separation speed is more important than resolution. The longer capillary is more effective for DNA sequencing where a longer size range with singlebase resolution is more desirable than rapid run times.

Capillaries used with the ABI 310 may or may not possess a covalently attached internal wall coating. Many capillaries used for CE are 'coated' by chemical derivatization to prevent a process known as electro-osmotic flow (EOF). Above pH 6, silica is negatively charged and thus the inside wall of a fused silica capillary will be covered by negative charges. EOF results from positive charges from the solution inside the capillary that form along the negatively charged capillary wall in what is known as the 'double layer'. Upon the application of an electric field, the mobile cations in the double layer migrate toward the cathode (inlet) and pull solution molecules in the same direction.

EOF is in the opposite direction as electrophoretic migration of the DNA molecules and thus slows their progression through the capillary. EOF is highly dependent on environmental parameters, such as pH, temperature, voltage, and buffer viscosity. Thus, in order to obtain reproducible separations of DNA, EOF is removed by coating the inner wall of the capillary. There are two primary methods for coating the inner wall of a capillary: chemical derivatization of the charged silanol groups or dynamic coating with a viscous polymer solution.

The capillaries typically used in the ABI 310 CE system are 'uncoated' meaning that the interior surface of the capillary does not have any chemical modifications to cover the charged silanol groups. However, the POP-4 polymer solution used for DNA separations dynamically coats the inside capillary wall and prevents EOF.

POLYMER SOLUTION

Applied Biosystems currently sells two polymer formulations for use with the ABI Prism 310 Genetic Analyzer. POP-4™ and POP-6™, which stands for Performance Optimized Polymer, are 4% and 6% concentrations of linear, uncross-linked dimethyl polyacrylamide, respectively. A high concentration of urea is also present in the polymer solution to help create an environment in the capillary that will keep the DNA molecules denatured.

POP-4 is a commercially available preparation of a flowable polymer [poly (A, A-dimethylacrylamide); DMA]. POP-4 contains 4% DMA homopolymer, 8M urea, 5% 2-pyrrolidinone, and 100mM A-Tris-(hydroxymethyl) methyl-3-aminopropane-sulfonic acid (TAPS) adjusted to pH 8.0 with NaOH (Rosenblum et al. 1997).

POP-4 is most commonly used for STR typing while POP-6, which is more viscous and yields improved resolution at the expense of longer run times, is typically used for DNA sequencing applications. To prepare the CE system for use, approximately 600 or 700 ||L are drawn up into a 1 mL syringe, which is placed in the ABI 310 instrument. Between 100 and 300 ||L are often needed to prime the CE system and drive out any air bubbles. Several microliters are used between each DNA separation to refill the capillary for the next run. A full syringe can therefore last for 100 or more unattended injections prior to needing a refill.

ELECTROPHORESIS BUFFER

The electrophoresis buffer supplies the ions for conducting current across the capillary. If it is not properly replenished, the current can fluctuate affecting the DNA separation. Applied Biosystems supplies a 10X Genetic Analysis buffer with EDTA that is typically used in STR sample separations. The user simply dilutes the 10X buffer with nine times the volume of water to make a 1X solution. The 1X concentration of the Genetic Analysis buffer is 100 mM TAPs, 1 mM EDTA, pH 8.0 (Rosenblum et al. 1997). The electrophoresis buffer should be replaced after every set of about 100 sample injections.

OPERATION OF THE ABI 310 FOR GENOTYPING STR SAMPLES

The sample processing steps using the ABI 310 are illustrated in Figure 14.2. Samples are prepared and loaded into an autosampler tray. The user then enters the sample names and positions into a Sample Sheet where comments concerning each sample may be entered along with the fluorescent dyes (blue, green, yellow, and red) in the sample. An internal sizing standard is also included in each sample and the appropriate color (usually red or yellow) needs to be indicated at this point.

Following the completion of the sample sheet, an injection list is then created and the sample sheet information imported into it. On the injection list, an operation module is selected for each sample. The typical module used for STR typing is 'GS STR POP4 (1 mL) F' which includes a default injection of 15 kV for five seconds and a separation voltage of 15 kV for 24 minutes.

SAMPLE PREPARATION

Following PCR amplification of forensic DNA samples using a commercially available STR typing kit, the resulting fluorescently labeled STR alleles need to be separated, sized, and genotyped. Samples are prepared for the ABI 310 by diluting them in a denaturant solution that helps disrupt the hydrogen bonds between the complementary strands of the PCR products. An internal standard is also added to each sample for sizing purposes. The samples are then heat

Replace capillary

Refill syringe with polymer solution

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