Detection And Data Analysis

In early separations of PCR products by CE, UV absorbance was the method of detection. PCR products analyzed by this procedure required extensive deionization and concentration prior to analysis (20,41,42). The relatively short path length and the dispersion produced by the capillary walls limited sensitivity. Laser-induced fluorescence (LIF) solved the problem of sensitivity by focusing the light beam directly onto the capillary window (36,47,48). Detection enhancements of 400-fold or more have been achieved using LIF as compared to UV detection (36).

Fig. 3. An illustration of the 45-s separation of two PCR products coinjected with a 20 basepair sizing ladder. The analysis was performed on a 2-cm coated capillary at 260 v/cm. The DNA was detected using an intercalating dye and laser-induced fluorescence (45).

Fluorescence detection of dsDNA is primarily achieved through the use of intercalating dyes. These dyes bind to the DNA molecule by inserting themselves into the DNA helix, affecting the configuration of the aromatic rings of the dye and enhancing the fluorescence signal (38,49). Additionally, intercalating dyes help to minimize effects of DNA structure on migration rate, resulting in better estimates of fragment lengths (39,50). The low background fluorescence of the uncomplexed dyes allows them to be added directly to the CE buffer. Monomeric intercalating dyes such as ethidium bromide, thiazole orange, and oxazole yellow have proven to be the most useful, providing precise and reproducible estimates of DNA size and quantity (40,51).

Fluorescent dye molecules may also be covalently bound to the DNA fragments (34). Labeling one or both of the primers prior to the amplification step can perform this most efficiently. After completion of the PCR, all of the target DNA molecules are labeled with a fluorophore. By labeling a series of different primers with a number of different dyes, several loci may be targeted, amplified, and labeled in a single multiplexed reaction. These dyes absorb at similar wavelengths but emit at different wavelengths. A multichannel analyzer can then identify the specific PCR product by detecting the various emission wavelengths of the bound dye (52).

The development of methods for data analysis by CE is of particular importance in the examination of PCR products. Precise and reliable meth-

Fig. 4. The relationship between DNA size and migration time for native DNA. The figure illustrates that a linear relationship exists up to 400 basepairs.

ods must be developed for product analysis. Slab gel methods permit the analysis of multiple samples run concurrently. At present, CE is a serial technique and samples can only be run one at a time. Thus, comparison of multiple samples requires the addition of internal standards to correct for the inevitable variations in injection, temperature, and current (22). This observation is particularly relevant in quantitative methods where variations in sample injection can limit the usefulness of the technique (40).

When adding internal standards for quantitation, it is important that they do not interfere with the detection of the PCR product. When intercalating dyes are used in the analysis, peak intensity is a function of the length of the PCR product, and corrections for product length may be necessary (53). A further concern in PCR analysis using these dyes is the effect of buffer depletion on the sample fluorescence intensity and migration time (40,50). For this reason, buffer vials must be periodically replenished to avoid depletion of the intercalating dye and pH variations.

Size estimates of PCR products can be performed by interpolation of size based on the migration of one or more internal standards (22). For products in the size range from 100-400 bp, a linear relationship exists between size and migration time (Fig. 4) (54). The size of larger products may also be estimated using nonlinear, curve-fitting algorithms (50). Specialized instrumentation has been developed specifically for DNA analysis utilizing internal standards that have been labeled with a fluorescence dye different than that of the product (52). For such systems, interferences between sample and standard are much less of a problem, and specific algorithms have been developed to deconvolute the fluorescence signals and perform size estimates (55).

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