Reliability of SE33 typing by capillary electrophoresis

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Abstract

A ladder of 24 ACTBP2 (SE33) alleles was separated 175 times by denaturing capillary electrophoresis on an ABI Prism™ 310 Genetic Analyzer using polymer POP-4™. The mean standard deviation of fragment size determination was 0.083 bp. Fragments in the whole allelic range of ACTBP2 could be typed with high precision and reproducibility if adjacent fragments differed by at least two nucleotides. The capacity of resolving 1 bp differences was tested by repeatedly running a ACTBP2*14.2/14.3/31.2/31.3 allelic mixture. The 14.2/14.3 fragment pair could be separated in 98%, the 31.2/31.3 fragment pair only in 65% of all runs. Reliable separation of this difficult fragment mixture could exclusively achieved by using POP-6™.

Introduction

Due to its tremendous variability ACTBP2 (SE33) is a powerful short tandem repeat (STR) for forensic DNA-analysis. The presence of numerous interalleles (#.1, #.2, #.3), however, makes high demands on the resolving capacity of the electrophoresis device used [1], [2], [3], [4], [5]. Capillary electrophoresis (CE) is commonly applied for DNA fragment length determination and sequencing. It shows high-precision and reproducible sizing operation, and therefore CE should fit the demands of such a complex STR system like ACTB2 [6], [7], [8], [9].

We investigated, how precise and reliable the ABI Prism™ 310 Genetic Analyzer determines the length of amplified SE33-fragments under standard conditions with special respect to the capacity of separating 1 bp-differences. Standard deviation of fragment lengths were obtained by repeatedly running an allelic ladder. An attempt was made, to find optimized electrophoretic conditions to increase the separation performance.

Section snippets

PCR-amplification and electrophoresis methods

A ladder of 24 sequenced SE33-alleles (Fig. 1), kindly given by A. Junge, Institute of Legal Medicine, University Bonn, Germany [3], [4], [5], was separated 175 times on a ABI Prism™ 310 Genetic Analyzer (PE Applied Biosystems) in a 5–47 cm×50 μm capillary (PE Applied Biosystems; Part No. 4028399) using performance optimized polymer POP-4™ and the corresponding module GS STR Pop 4 with 26 min separation time. Alleles were sized running the GeneScan™ 2.1 software (ROX350™ size standard; local

Results and discussion

The mean value for the standard deviation (σ) was 0.083 bp (min=0.071 bp; max=0.093 bp; Table 2). 99.62% (=4184) of the 4200 measured SE33 alleles were within the ‘±3σ’-interval. 0.38% (=16) showed slightly larger sizes, but were still within the ‘+4σ’-interval. These were observed within three runs showing a general small shift towards larger calculated allele sizes. The results obtained here are comparable to those of other authors using capillary electrophoresis [6], [7], [9] or the ABI

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