Design, installation, and performance evaluation of a custom dye matrix standard for automated capillary electrophoresis

Abstract

CE equipment detects and deconvolutes mixtures containing up to six fluorescently labeled DNA fragments. This deconvolution is done by the collection software that requires a spectral calibration file. The calibration file is used to adjust for the overlap that occurs between the emission spectra of fluorescence dyes. All commercial genotyping and sequencing kits require the installation of a corresponding matrix standard to generate a calibration file. Due to the differences in emission spectrum overlap between fluorescent dyes, the application of existing commercial matrix standards to the electrophoretic separation of DNA labeled with other fluorescent dyes can yield undesirable results. Currently, the number of fluorescent dyes available for oligonucleotide labeling surpasses the availability of commercial matrix standards. Therefore, in this study we developed and evaluated a customized matrix standard using ATTO 633, ATTO 565, ATTO 550, ATTO Rho6G, and 6-FAM dyes for which no commercial matrix standard is available. We highlighted the potential genotyping errors of using an incorrect matrix standard by evaluating the relative performance of our custom dye set using six matrix standards. The specific performance of two genotyping kits (UniQTyper™ Y-10 version 1.0 and PowerPlex® Y23 System) was also evaluated using their specific matrix standards. The procedure we followed for the construction of our custom dye matrix standard can be extended to other fluorescent dyes.

Allelic ladder production for a 10 locus Y‐chromosome DNA profiling system

“…This is a significant difference given that all PCR template input concentrations were normalized. This superior signal at DYS644 and DYS385 alleles (not shown) was previously noted during the construction of the dye matrix [21]. This may be attributed to the high fluorescence quantum yield, and high thermal and photo‐stability known for the ATTO Rho6G fluorophore [23].…”

“…The primer sequences used for singleplex PCR were previously published in refs. [16,18] for which a customized fluorescence panel was developed [21]. The singleplex PCR amplifications were conducted using the master mix of the UniQTyper™ Y‐10 kit [18,19] containing Taq polymerase and 0.2 μM (forward and reverse) primer in a 25 μL PCR reaction.…”

“…A total of 1 μL PCR product was injected on an ABI3500 (Thermo Fisher) and spectral calibration achieved using a custom five‐dye matrix standard [21]. Samples were prepared using 9.7 μL HiDi™ formamide (Thermo Fisher), 0.3 μL GSLIZ 500 (Thermo Fisher).…”

Automated detection and removal of capillary electrophoresis artifacts due to spectral overlap

“…CE is currently the primary DNA detection method for both Sanger sequencing and DNA‐based fragment analyses and has been the primary method of DNA detection in the forensic human identification field since the late 1990s . This method has two primary advantages over other detection methods: it affords the ability to achieve single base resolution, and it permits the use of multi‐dye PCR multiplexes with up to six dye channels . These features are particularly advantageous when used for human identification and the CODIS loci where alleles may differ by a single base pair and higher levels of discrimination are achieved through the use of increased numbers of loci using multiple dye sets.…”

Allele Size Miscalling due to the Pull-Up Effect Influencing Size Standard Calibration in Capillary Electrophoresis: A Case Study Using HEX Fluorescent Dye in Microsatellites

“…In addition, because allele calling from capillary electrophoresis depends on detecting the emission spectra of fluorescent dyes, a spectral standard is needed to compensate for the emission spectrum overlap between dyes. Therefore, an incorrect spectral standard will also cause the pull-up effect [8].…”