Use of matrix standards for new fluorophores in capillary sequencers

Abstract

Most laboratories presently carrying out forensic identification and parentage testing use capillary DNA sequencers to separate and visualize the alleles that are produced by multiplex PCR amplification, whether they be of STR, SNP or DIP loci. Because at least four, but usually five, different fluorophores are used simultaneously, each instrument must undergo its own spectral calibration in which a matrix is created and used to correct the overlapping of fluorescence emission spectra of the mixture of dyes. For this users must acquire matrix standards in addition to the multiplex PCR kits and it is not clear what these standards consist of. In our efforts to develop new multiplex STR and DIP amplification kits using different fluorophores from those commonly used, we were obliged to produce new matrix standards to calibrate our multicapillary sequencers, and we describe how this was done.