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The AmpFISTRÔ Profiler PCR Amplification Kit coamplifies nine STR loci and amelogenin, a gender-determining marker. The primers for each of the loci are 5'-labeled with one of three fluorescent dyes and a fourth dye is used for the in-lane size standard. Following excitation with an argon ion (blue) laser, the fluorescent dyes are detected on a CCD camera on either the ABI PRISMÔ 310 Genetic Analyzer or the ABI PRISMÔ 377 DNA Sequencer. The AmpFISTRÔ Profiler kit utilizes a new dye set called "F", which includes the NHS-ester dyes 5-FAM, JOE, NED, and ROX. The advantages of this dye set are that these dyes have similar sensitivities and that the spectral resolution is improved relative to other dye sets. This new dye set requires new module files which specify the corresponding F virtual filter set. The F filter and dye sets will be presented and compared to the A and C filter and dye sets.

While the filter set F dyes exhibit four distinct emission maxima, spectral overlap still exists, and therefore, the use of a multicomponent matrix is necessary. The creation and application of a matrix will be explained. In addition, matrix troubleshooting will be discussed. This will include examples of accurate and inaccurate matrices and a characterization of "pull-up" (bleed-through peaks) and poor baselining.

While the accuracy of the matrix is important in achieving successful multicomponent analysis, the "symptoms" of an inaccurate matrix (pull-up and poor baselining) may also be observed when peaks are "off-scale". An off-scale peak results when the fluorescence intensity from the sample saturates the CCD camera detector. Pull-up will be observed because the actual peak height for the off-scale peak cannot be determined, and thus, accurate multicomponent analysis cannot be performed. Methods to identify on-scale and off-scale peaks on the ABI PRISMÔ 310 Genetic Analyzer and the ABI PRISMÔ 377 DNA Sequencer will be explained.

In casework evidence in which there are mixtures of DNA, the quantitation of stutter peaks and heterozygous peaks of a single locus (peak height ratios) can be useful in assigning genotypes to each contributor. In performing quantitative determinations, therefore, it is extremely important that the peak height quantitation is accurate. Only peaks that are detected within the linear dynamic range of the detector on the instrument (on-scale peaks) should be used in such calculations. Examples will be presented which demonstrate lack of quantitative accuracy when the data is off-scale, thereby affecting interpretation of results. When an accurate matrix is applied to peaks that are on-scale, successful multicomponent analysis is achieved for AmpFISTRÔ Profiler results and the quantitative information can aid in the interpretation of data.