The world is full of obvious things, which nobody by any chance ever observes.
(Sherlock Holmes, The Hound of the Baskervilles)
This chapter will examine several electrophoresis instrumentation platforms commonly utilized for analysis of STR loci: the single capillary ABI Prism 310 Genetic Analyzer, the 16-capillary ABI Prism 3100, and the FMBIO Fluorescence Imaging System.
Since its introduction in 1995 by Applied Biosystems, the ABI Prism 310 Genetic Analyzer has been an increasingly popular method for short tandem repeat (STR) typing in forensic DNA laboratories. A vast majority of forensic DNA laboratories within the United States use the ABI 310 for performing STR genotyping (Steadman 2000). In addition, the FBI Laboratory in Washington D.C. performs its STR typing with the ABI 310.
The ABI 310 is a single capillary instrument with multiple color fluorescence detection that provides the capability of unattended operation. An operator simply loads a batch of samples in the 'autosampler,' places a capillary and a syringe full of polymer solution in the instrument, and starts the 'run.' The data and genotype information are serially processed at the rate of approximately one sample every 30 minutes of operation. A major advantage of the technique for forensic laboratories is that the DNA sample is not fully consumed and may be retested if need be.
Many forensic scientists are using the ABI 310 without background knowledge of capillary electrophoresis (CE). This chapter reviews the theory and practice of capillary electrophoresis with a particular focus on the capabilities of the ABI Prism 310 Genetic Analyzer for genotyping STR markers (Butler et al. 2004). A few troubleshooting tips for the ABI 310 are also included (see McCord 2003).
The first CE separations of STR alleles were performed in late 1992 using non-denaturing conditions with the polymerase chain reaction (PCR) products in a double-stranded form (McCord et al. 1993a, 1993b). Fluorescent intercalating dyes were used to visualize the DNA and to promote the resolution of closely spaced alleles. Internal standards were used to bracket the alleles in order to perform accurate STR genotyping. An allelic ladder was first run with the internal standards to calibrate the DNA migration times followed by analysis of the samples with the same internal standards (Butler et al. 1994). This internal sizing standard method involving a single fluorescent wavelength detector had to be used because multiple color fluorescence CE instruments were not yet available. Since the commercialization of the ABI 310, internal standards labeled with a different color compared to the STR alleles can be used to perform the DNA size determinations and subsequent correlation to obtain the STR genotype.
Early on in the development of CE for DNA separations, one of the major concerns included sample preparation. PCR-amplified samples had to be dia-lyzed to remove salts that interfered with the injection of DNA fragments onto the CE column in order to observe the DNA with an ultraviolet (UV) detector. With the higher sensitivity of laser-induced fluorescence, sample preparation is no longer a major concern but does still play a role. Samples may be diluted in water or formamide and easily detected.
As discussed in Chapter 12, capillary electrophoresis involves the use of a narrow capillary filled with a polymer solution instead of a gel to perform the DNA size separation. The higher surface area-to-volume ratio in a capillary permits more efficient heat dissipation generated by the electrophoresis process and thus enables a higher separation voltage to be applied. Typical DNA separation times using CE are in the range of 5-30 minutes, compared to several hours for gel-based systems, because a higher voltage may be used. Most ABI 310 methods involve a separation voltage of 15 000 volts with a capillary length of 47 cm or 319 V/cm.
Polymer solutions have greatly aided DNA separations in capillaries. Prior to the injection of each new sample, a fresh portion of polymer solution is pumped into the capillary. This operation is analogous to pouring a new 'gel' automatically before each sample is loaded on the gel. The type and concentration of polymer solution used determines the resolution that may be obtained much in the same way that the percentage of cross-linking in polyacrylamide gels reflects the resolution capabilities of the electrophoretic system.
While CE is rapid on a per sample basis, it is a sequential technique where only one sample is analyzed at a time and is not as useful when trying to process large numbers of samples in parallel. Therefore, throughput is on the same time scale as, or even slower than, conventional gel electrophoresis methods.
As will be discussed in a future section, capillary array systems with 16 or 96 capillaries in parallel have been developed to aid in high-throughput operations.
DNA samples are loaded onto the capillary by applying a fixed voltage for a defined period of time or by applying pressure to the sample and forcing a plug of sample to enter the inlet end of the capillary. In the case of the ABI 310, only the voltage application or 'electrokinetic' injection mode is available for injecting DNA samples.
The basic components of the ABI Prism 310 Genetic Analyzer are illustrated in Figure 14.1. A capillary is located between the pump block and the inlet electrode. The capillary is filled with polymer solution through the pump block. A heated plate is used to heat the capillary to a specified temperature. Samples are placed in an autosampler tray that moves up and down to insert the sample onto the capillary and electrode for the injection process.
Prior to running any samples, the ABI 310 CE system must be readied for analysis. This preparation generally involves four steps:
1. Putting a capillary in the instrument and aligning the detection window;
2. Loading the polymer solution into a syringe and priming the system to remove any bubbles;
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