Laser excitation 532 nm

Figure 13.6 Example of a matrix file table from an ABI Prism 310 instrument. These values are used by the GeneScanĀ® Analysis Software to separate the various dye colors from one another. The letters B, G, Y, and R represent the dye colors Blue, Green, Yellow, and Red, respectively. All matrix files should have values of 1.0 on the diagonal from top left to bottom right. The other values in the table should all be less than 1.0. These values represent the amount of spectral overlap observed for each dye in each virtual detection filter. Matrix file values differ between instruments and even run conditions on a single instrument. Thus, a unique matrix file must be made for an instrument and a particular set of run conditions.

Reactions B G Y R

B

1.0000

0.8502

0.1380

0.0009

G

0.8300

1.0000

0.7622

0.0051

Y

0.6416

0.8324

1.0000

0.1102

R

0.4493

0.6484

0.7851

1.0000

An example matrix from an ABI Prism 310 instrument may be seen in Figure 13.6. The values in this table represent the amount of spectral overlap observed for each dye in each color: blue (B), green (G), yellow (Y), and red (R). In the case of AmpFTSTR kits, the four fluorescent dyes are 5-FAM, JOE, NED, and ROX (Figure 13.4). Note that in the matrix file table (Figure 13.6), there are values of 1.0 on the diagonal from top left to bottom right and that all of the other values in the table are less than 1.0. These values represent the amount of spectral overlap observed for each dye. For example, the values in the B column are 1.000 (B), 0.8300 (G), 0.6416 (Y), and 0.4493 (R). Thus, in this example the most significant overlap is green into blue because 83% (0.8300) of the blue signal is made up of green on a normalized scale. Note that the emission spectra shown in Figure 13.4 also possess the most overlap between the blue and green dyes.

Matrix files differ between instruments and even different run conditions on the same instrument because fluorescence of a dye is affected by the dye's environment. If the environmental conditions, such as temperature, pH, etc., change even ever so slightly, then the fluorescence behavior of the dyes will be altered. A matrix file should therefore be generated frequently to ensure good dye color separation and certainly anytime instrument conditions are altered, such as running samples in a different buffer system. As long as the electrophoresis conditions are constant from run to run, then the emission spectra of the dyes will be reproducible and spectral overlap can be accurately deciphered.

If the matrix color deconvolution does not work properly than the baseline can be uneven or a phenomenon known as 'pull-up' can occur. Pull-up is the result of a color bleeding from one spectral channel into another, usually

(a) Data collected by instrument (colors not separated yet)

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