Developmental validation of GlobalFiler\u2122 PCR amplification kit: a 6-dye multiplex assay designed for amplification of casework samples

Matthew J Ludeman,Chang Zhong,Dennis Y Wang,Robert E Lagacé,Marc L Short,Lori K Hennessy,Julio J Mulero

doi:10.1007/s00414-018-1817-5

Abstract

The GlobalFiler™ PCR Amplification Kit is a single multiplex assay that amplifies a set of 24 markers, which encompass the European Standard Set and CODIS (Combined DNA Index System) recommended composite set of loci. In addition to more loci and a 6-dye chemistry format, the Master Mix has been formulated to allow higher sample loading volume for trace DNA samples. The GlobalFiler™ Kit has been optimized to deliver high performance on casework samples, while also delivering fast thermal cycling, with an amplification time of approximately 80 min. Here, we report the results of the developmental validation study which followed the SWGDAM (Scientific Working Group on DNA Analysis Methods) guidelines and includes data for PCR-based studies, sensitivity, species specificity, stability, precision, reproducibility and repeatability, concordance, stutter, DNA mixtures, and performance on mock casework samples. The results validate the multiplex design as well as demonstrate the kit’s robustness, reliability, and suitability as an assay for human identification with casework DNA samples.

Highlights

Genotyping by targeting short tandem repeats (STRs) present in the human genome has proven to be an extremely discriminating method for human identification in forensic and paternity applications for decades [1]
Once optimal conditions were identified, the robustness of the GlobalFilerTM Kit was examined by varying the times and temperatures over a relevant range in reactions using 1.0 ng of Control DNA 007
In amplifications where the denaturing temperature was varied by +/− 1.5 °C, profiles returned showed no apparent effect on performance

Summary

Introduction

Genotyping by targeting short tandem repeats (STRs) present in the human genome has proven to be an extremely discriminating method for human identification in forensic and paternity applications for decades [1]. With an ever-growing number of STR profiles being generated globally, there has been great interest in ensuring that methods are available to maintain high levels of discriminatory power to reduce the likelihood of adventitious matches, and to enable agencies to share information internationally as effectively as possible [2,3,4,5]. This is reflected in the expansion of the CODIS. We report findings that demonstrate the robustness of the GlobalFilerTM chemistry and its suitability for use in the

Methods

Results

Conclusion