Forensic Science International: Genetics Supplement Series 2 (2009) 119–120
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Research article
Validation of a microchip electrophoresis system as a DNA amplification control Bram Bekaert a,*, Monique Coomans a, Katleen Knaepen a, Lucie Larno a, Nathalie Thijs a, Elisabeth Vanhoutte a, Wim Van de Voorde a, Ronny Decorte a,b a b
UZ Leuven, Department of Forensic Medicine, Laboratory of Forensic Genetics and Molecular Archaeology, Kapucijnenvoer 33, B-3000 Leuven, Belgium K.U.Leuven, Department of Human Genetics, Campus Gasthuisberg, Herestraat 49, B-3000 Leuven, Belgium
A R T I C L E I N F O
A B S T R A C T
Article history: Received 14 August 2009 Accepted 14 August 2009
As part of the normal procedure in a forensic DNA laboratory, a quality control step of the amplified DNA is often implemented to ensure the correct amplification of the sample before it is analysed in downstream applications. A validation study was undertaken to investigate a new microchip electrophoresis system (MultiNa, Shimadzu Corporation) claiming high resolution and sensitivity compared to routine polyacrylamide gel electrophoresis (PAGE). An array of STR multiplexes (AmpFISTRTM SGM+, GenePrint1 FFFL, PowerPlexTM 16, PowerPlexTM Y, an in-house Y-STR multiplex and AmpFISTRTM Profiler) was tested under both standard and low copy number PCR parameters to evaluate the accuracy, reproducibility and sensitivity of this technique. These tests showed that the microchip system did not have improved sensitivity compared to PAGE though had increased resolution and high reproducibility between samples. ß 2009 Elsevier Ireland Ltd. All rights reserved.
Keywords: MultiNa Microchip electrophoresis Amplification control
1. Introduction The evaluation of the quantity and quality of amplified products, whether they are from mitochondrial or nuclear DNA, is an important step within a forensic genetics laboratory which helps to determine the required volume for downstream analyses. This evaluation is commonly performed by polyacrylamide gel electrophoresis, which is a laborious and hazardous technique when used in combination with ethidium bromide. In the present study, the MultiNa Electrophoresis System from Shimadzu Corporation was validated for routine evaluation of amplified mitochondrial and nuclear DNA. The technique uses up to four microchips in parallel for the electrophoresis of DNA or RNA samples in an 8-strip or 96-well plate format. The method of detection is by SYBRGold incorporation and results are displayed as a digital gel and electropherogram. An internal marker consisting of a lower and upper marker serves for inter-chip normalisation and a 25 bp ladder is used for sizing the fragments. The validation study aimed to determine the accuracy, sensitivity and reproducibility of the method for the evaluation of mitochondrial and nuclear DNA amplification products. 2. Materials and methods DNA extracts quantified by QuantifilerTM from three individuals was submitted to PCR in quadruplicate using AmpFISTRTM SGM Plus, GenePrint1 FFFL, PowerPlexTM 16, PowerPlexTM Y, an * Corresponding author. E-mail address:
[email protected] (B. Bekaert). 1875-1768/$ – see front matter ß 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.fsigss.2009.08.092
in-house Y-STR multiplex and AmpFISTRTM Profiler STR multiplex kits for nuclear DNA analysis while the hypervariable region HV1 was amplified from the mitochondrion [1–3]. DNA extracts were serially diluted from 2 ng to 0.125 ng and amplified using the recommended number of PCR cycles while dilutions ranging from 0.125 ng down to 0.015 ng were amplified using low copy number PCR settings (increased cycle number (32) and injection time settings). All microchip results were compared to standard PAGE results and as a final step two of all technical replicate dilution series were analysed on an ABI3130XL genetic analyser. 3. Results The accuracy of the instrument was determined by comparing the length of the amplified HV1 region. A mean amplicon length of 463 bp (SD 1.8 bp, min. 460 bp, max. 467 bp) indicated a 1% mean overestimation of the expected 459 bp, which was within the 5% range set by the manufacturer. Observed microchip HV1 peak heights were compared with nuclear DNA concentration and correlated very strongly (R2 = 0.99). Sequencing by ABI3130 revealed that samples with MultiNa peak height below 25 mV did not produce a signal high enough to allow the interpretation of the sequence. Reproducibility of the technique was evaluated by comparing the mean of the microchip peak heights from STR amplification products between four technical replicates (R2 = 0.96). A subjective comparison of band intensity between PAGE and microchip digital gel pictures suggested no increased sensitivity for the SYBRGold method compared to the ethidium bromide method. The samples were subsequently analysed on the ABI3130XL genetic analyser and
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B. Bekaert et al. / Forensic Science International: Genetics Supplement Series 2 (2009) 119–120
Fig. 1. Output generated by the MultiNa Viewer software. (A) Digital gel of an AmpFISTRTM SGM+ amplified dilution series (pg). 2000–125 pg amplified with 28 cycles, 125– 15 pg amplified with 32 cycles; (B) electropherogram of a 2 ng sample amplified with AmpFISTRTM SGM+.
the % profile recovered was compared to the number of peaks obtained on the microchip system. A correlation between DNA profile recovery rate and microchip peak number or height was poor. Results show that even without the presence of a single allelic peak on the microchip system, a full profile could be recovered after analysis on ABI3130XL. On the other hand, a full DNA profile was always obtained when 10 MultiNa peaks or more were observed. This constant was used as a threshold below which secondary measures should be taken (such as increased volume and injection time on the genetic analyser) to increase allele detection. In summary, the validation experiment showed that (i) the microchip system does not have increased sensitivity compared to the PAGE method and is therefore not sensitive enough to be used as a general DNA profile success predictor; (iii) the accuracy and reproducibility are equal to or higher than expected values. 4. Discussion and conclusion The recovery of full DNA profiles when no peaks were observed with the microchip system proved it was not able to discriminate between samples that would yield a positive or negative result after analysis. This is due to the fairly low sensitivity of the instrument,
which was unexpected. The advantages of the microchip system, however, are the quantification of the amplification products, accurate sizing of the fragments, the presentation of the results in a digital gel/electropherogram format (Fig. 1), the reduction in labour time (approximately 30 min to set up a full plate and prepare the instrument) and a lower volume requirement for the analysis (5 mL in the 96-well format vs. 10 mL for PAGE). Conflict of interest None References [1] R. Decorte, M. Engelen, L. Larno, K. Nelissen, A. Gilissen, J.J. Cassiman, Belgian population data for 15STR loci (AmpFISTR SGM Plus and AmpFISTR Profiler PCR amplification kit), Forensic Sci. Int. 139 (2004) 211–213. [2] K. De Maesschalck, E. Vanhoutte, K. Knaepen, N. Vanderheyden, J.J. Cassiman, R. Decorte, Y-chromosomal STR halotypes in a Belgian population sample and identification of a micro-variant with a flanking site mutation at DYS19, Forensic Sci. Int. 152 (2005) 94–98. [3] R. Decorte, E. Verhoeven, E. Vanhoutte, K. Knaepen, J.J. Cassiman, Allele frequency data for 19 short tandem repeats (Powerplex 16 and FFFL) in a Belgian population sample, J. Forensic Sci. 51 (2006) 436–437.