Elsevier

International Congress Series

Volume 1239, January 2003, Pages 901-904
International Congress Series

Validation and practical experiences with the multiplex kits genRES® MPX-2 (SERAC) and GenePrint® Powerplex™ 16 (promega)

https://doi.org/10.1016/S0531-5131(02)00511-3Get rights and content

Abstract

Validation studies were carried out using the commercially available multiplex systems GenePrint® PowerPlex™ 16 and the genRES® MPX-2. In a first approach, sensitivity and mixture studies were investigated with quantified human DNA. The minimum amount of template DNA that gave a complete DNA pattern ranged between 200 and 500 pg for both multiplex systems. The mixture ratios that were detectable and could be clearly assigned were determined to be 1:5/1:10. In order to get more experience, three pieces of fresh muscle tissue and a 6-month-old microbloodstain on a pacemaker were investigated. The preliminary results of the tissue study revealed that the MPX-2 amplification pattern is more homogeneous than PowerPlex16 and that PowerPlex16 is less robust towards template DNA variation compared to MPX-2 resulting in the increased occurrence of imbalanced peak heights and locus drop-out. As shown in the case of the 6-month-old microbloodstain, the age of the stain material seems to be more crucial for PowerPlex16 than for MPX-2 amplification.

Introduction

In the past, a number of different multiplex systems have been validated and established for forensic identification purposes and are now commercially available. The aim of the establishment was to reduce time, cost and material, and to maximize the forensic power of discrimination. At the beginning of the multiplex era, the systems consisted of no more than three short tandem repeat (STR) systems [1], [2]. Meanwhile, the number of systems that can be amplified in a single reaction has increased up to 15 STR systems [3], [4], [5]. Before application of multiplex systems in routine casework, extensive validation studies have to be performed, e.g. sensitivity and mixtures studies and experimental stain casework, in order to get information about reproducibility, robustness and specificity. Validation studies were carried out with the commercially available multiplex kits genRES® MPX-2 and GenePrint® PowerPlex™ 16. The genRES® MPX-2 kit contains eight STRs (TH01, VWA, FGA, ACTBP2, D21S11, D3S1358, D8S1179, D18S51) which are components of the German DNA database and the sex-specific Amelogenin. The GenePrint® PowerPlex™ 16 kit co-amplifies 15 STRs+Amelogenin including the 13 CODIS tetranucleotide STR loci and 2 pentanucleotide STR loci. The aim of the study was to perform sensitivity and mixture studies with defined amounts of template DNA and to validate the multiplex systems for forensic casework.

Section snippets

Material

The sensitivity and mixture studies were carried out with quantified human DNA. The case work studies included blood and saliva samples, microbloodstains and tissues samples.

DNA extraction

DNA was extracted using Chelex® 100 for blood and saliva samples or the All-tissue DNA-Kit (GEN-IAL, Troisdorf, Germany) for tissue samples.

Sensitivity and mixture studies

Sensitivity studies were carried out with different amounts of quantified human DNA ranging from 5 to 0.05 ng. The mixture ratios were 1:1, 1:2, 1:5, 1:10, 1:20, 1:50 and 1:100 with the

Sensitivity and mixture studies

Sensitivity studies revealed that for both the MPX-2 and the PowerPlex16 kit (Fig. 1), all alleles could be clearly detected and assigned to the allelic ladder at levels between 500 and 200 pg template DNA (Table 1). The phenomenon of allelic drop-out/preferential amplification was observed applying DNA levels below 200 pg. As expected, the sensitivity of the multiplex systems is lower than that of the singleplex amplification. In studies with different mixing ratios and the constant component

Conclusions

Both multiplex systems showed similar results concerning sensitivity and mixture ratios. As expected, the multiplex systems are less sensitive (200–500 pg) than loci amplified in a singleplex reaction. In general, blood and saliva samples could be typed without problems. Insufficient amplification of ACTBP2 (MPX-2) and of the loci with longer fragment lengths (PowerPlex16) was partly observed. Preliminary results of our investigations on forensic material showed that the MPX-2 amplification

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