Wavelength nm

Laser excitation (488, 514.5 nm)

310 Filter Set F with color contributions

Each dye set used must be matched to the instrument optics involved in detection as well as the excitation source. For example the ABI Prism 310 uses an Argon ion (Ar+) laser with excitation wavelengths at 488 nm and 514nm while the FMBIO II uses a solid-state Nd:YAG laser with an excitation wavelength of 532 nm. PowerPlex® 1.1 fluorescein-labeled primers, designed for FMBIO detection, are present in higher concentration than the same primers in PowerPlex® 1.2, designed for ABI 310 detection, because of the different laser excitation wavelengths. Because the FMBIO laser is not as well suited for fluorescein dye excitation, more primer must be put in the PCR reaction to generate balanced product amounts compared to TMR-labeled amplicons.

When labeling DNA fragments with various dyes it is important to use appropriate concentrations of the dyes in order to obtain balanced signals between loci. For example, because NED has an excitation maximum that is further from the Ar+ 488/514nm lines more dye is required in order to obtain an equivalent signal to FAM.

Figure 13.5 Schematic of three-color detection using the Hitachi FMBIO Scanner System. These dyes and spectral filters (shown with boxes) are used for detection of STR alleles amplified with the Promega PowerPlex systems.

ISSUES WITH FLUORESCENCE MEASUREMENTS

Multi-component analysis is performed with a mathematical matrix that subtracts out the contribution of other dyes in each measured fluorescent dye. 'Color deconvolution' is another phrase to describe what this matrix does. When raw data is collected from a fluorescence-based detection platform, spectral overlap of different dyes must be accounted for in order to gain the capability to see the results from each dye individually.

One method of performing multi-component analysis is to examine a standard set of DNA fragments labeled with each individual dye, known as matrix standard samples (Applied Biosystems 1998). Computer software then analyzes the data from each of the dyes and creates a matrix file to reflect the color overlap between the various fluorescent dyes. This matrix file table contains a table of numbers with four rows and four columns if there are four dyes that are being deconvoluted. Alternatively, a 5 X 5 matrix is generated with 5-dye chemistries on the ABI Prism 310 Genetic Analyzer.

fluorescein TMR CXR

505 585 650

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