S0 200 220 240

| - Repmaenti md knr dy* égral for standard ladder.

| | • Represents Mus ta«*r dye «Ignal tor PCR products

Fig. 5. Dual wavelength detection of a multiplex amplified PCR product consisting of three genetic loci, D351358, vWA, and FGA. Conditions: 3% HEC, 7 mM urea, 100 mM Tris-borate, pH 8.4, 375 V/cm. The amplified product is labeled with a blue emitting dye, and the internal sizing standard is labeled with a red emitting dye.

obtained from the multiplex amplification of four different loci labeled with a blue-emitting dye. The sample is diluted in purified formamide and denatured. An internal standard labeled with a red-emitting dye is added to each sample to estimate fragment size (30,62). The samples are then analyzed using a viscous buffer containing a soluble polymer in addition to 7 M urea to keep the sample DNA denatured. The analysis temperature is kept at 60°C to melt out (eliminate) secondary structures that can cause problems with reproducibility.

5.2. Analytical Aspects in Developing a CE Separation

Among the instrumental factors that are important in developing a CE assay for genetic typing, precision and resolution are perhaps the most important. These two issues, however, are not dependent on each other. Precision is determined by the run to run reproducibility of the migration time of the peak apex, whereas resolution is a function of band broadening and column efficiency.

The precision of the assay is important because it defines the minimum difference in allele size that can be determined. For example, if the size of a particular allele is 200 bases with a standard deviation of 0.17 bases, then 99.3% of the time that allele will be given a size of 200 bases, assuming a normal distribution of the data. Such precision is important given the existence of variant alleles that can differ from the normal 4 base repeat motif by 1-3 bases. In addition, many forensic samples are mixtures of more than one donor.

The mixture problem also can be a factor when resolution is considered. In the aforementioned example, the precision of 0.17 bases permits us to distinguish between a peak at 200 and 201 bases. However if the resolution,

between the two peaks is 0.67 or less, it will be difficult to resolve these two peaks. If the area of peak 1 is more than three times that of peak 2, the two peaks will appear to co-elute, as the system will not have the capacity to separate the two peaks (63). This is illustrated in Fig. 6.

Thus, when developing a protocol for the analysis of mixtures by CE, it is important to consider both precision and resolution. As mentioned earlier, the

Fig. 6. (opposite page) The analysis of a mixture of two samples of PCR amplified DNA. The large peak at the right is a mixture of two DNA samples, which measure 185 and 186 bases in length. At a ratios from 20:1 to 3:1 the software is unable to distinguish the fact that two peaks exist. At ratios of 1.4:1 and 1:1, the peak to the right can be distinguished and is colored gray. Conditions: ABI310 Capillary electrophoresis system, POP4 buffer system 15 kV.

Fig. 6

precision is determined by factors that affect the stability of the measurement; temperature, injection, and sample conformation. The resolution is primarily affected by the polymer concentration and by the effects of sample stacking on injection. Both factors must be characterized to achieve optimum results.

A third issue that can be of importance is the determination of peak area. In situations where mixtures are present, the peak area can help define which samples are related. With proper control of the PCR conditions, the areas of related sample alleles will be consistent from one locus to the next, allowing the user to determine major and minor contributors to the electrophoretic profile. There are, however, important exceptions to this rule, which arise from the nature of the PCR reaction and from the injection process. Issues in the PCR process include artifacts such as stutter, in which a minor product band is produced one repeat unit shorter than the main allele. Values of stutter of 4-9% were reported for the vWA locus (64). The presence of stutter can affect the areas of peaks by decreasing the efficiency of amplification of the main peak, and by interfering with the areas of nearby peaks. PCR efficiency can also be affected by low quantities of template and improper reaction conditions. Another factor to consider is matrix effects on sample injection. The sample matrix can influence the overall peak area from one run to the next, however, this effect can be corrected through proper reference to the internal standard.

At present there have been a number of reports in the literature regarding the analytical capability of capillary systems for typing STRs (30,62,65,66). At least three different polymer systems have been reported for multiplex PCR analysis: hydroxyethyl cellulose, polydimethyl acrylamide, and linear polyacrylamide. Resolution varies between 1 and 2 bases depending on the size of the allele and the concentration and type of the polymer. Reported precision of the estimated allele size as measured by standard deviation ranges from 0.16-0.23 bases. These results clearly show that 2 base differences will be easily distinguished by these systems and depending on the allele size and polymer type, single-base differences may also be distinguished. In an extensive forensic validation study, Wallin and coworkers demonstrated and compared the results on the validation of the AmpliSTR Blue locus using both slab gels and CE (80). In this work, population samples from different racial groups were examined as well as studies on the effect of environment, sample matrix, and sample mixtures. Figure 7 illustrates results from this paper in which two nonprobative sexual assault cases were analyzed by CE using the POP4 polymer. The results portray an exclusion of the suspect in the first case: however, in the second case suspect 2 is a potential contributor of the sample DNA.

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