Electrophoresis 2012, 33, 2833–2839 2833 Zahra Nathalie ∗ Sibte Hadi William Goodwin School of Forensic and Investigative Sciences, University of Central Lancashire, Preston, UK Received April 30, 2012 Revised June 14, 2012 Accepted June 14, 2012 Research Article Development of PCR internal controls for DNA profiling with the AmpFℓSTR R  SGM Plus R  amplification kit Forensic DNA profiling uses a series of commercial kits that co-amplify several loci in one reaction; the products of the PCR are fluorescently labelled and analysed using CE. Before CE, an aliquot of the PCR is mixed with formamide and an internal lane size standard. Using the SGM Plus amplification kit, we have developed two internal non-amplified controls of 80 bp and 380 bp that are labelled with ROX fluorescent dye and added to the PCR. Combined with two internal amplification controls of 90 bp and 410 bp, they provide additional controls for the PCR, electrokinetic injection, and CE and also function as an internal size standard. Keywords: Forensic / IACs / Internal amplification controls / PCR inhibitors / SGM Plus R  DOI 10.1002/elps.201200252 1 Introduction Robust interpretation of DNA profiles generated by kits such as the AmpFℓSTR R  SGM Plus R  depends on the quality of the DNA samples, amplification efficiency and the success of post-PCR processing. Poor quality template that is degraded or low copy number can give a weak or partial signal while chemicals often co-extracted with DNA can inhibit the ampli- fication process [1–3]. Furthermore, random errors occurring during pipetting and electrokinetic injection lead to signal variability. Depending on the degree of the effect, these fac- tors can pose a challenge in evaluating DNA profiles. Various assays have been developed to detect the pres- ence of PCR inhibitors and most of these include the use of internal amplification controls (IACs or internal PCR con- trols). These are non-target DNA fragments added to the sam- ple and co-amplified with the target DNA [4–7]. The presence of the IAC signal indicates success of the PCR even when no target DNA is present, whilst a reduction of the maker demonstrates a problem in the amplification process. Commercial kits such as Quantifiler R  kit [8] and Plexor R  qPCR [9] include an IAC in their quantitative PCR to check for inhibitors. Wurmb Schwark [10] also described the use of separate amplification reaction to assess the quality of the DNA prior to the profiling reaction using a 271 bp fragment as an IAC. Previously, we presented the development of IACs Correspondence: Dr. William Goodwin, Reader in Forensic Genet- ics, School of Forensic and Investigative Sciences, University of Central Lancashire, Preston, PR1 2HE, UK E-mail: whgoodwin@uclan.ac.uk Fax: +44-1772-894981 Abbreviations: IAC, internal amplified control; INAC, internal non-amplified control; ISS, internal size standard; PIC, PCR internal control with amplicon sizes of 90 bp and 410 bp that were added directly to the AmpFℓSTR R  SGM Plus R  reaction, allowing the direct assessment of PCR efficiency without using an additional kit or DNA material that in casework samples may be limited [11]. Other factors that often affect the profile signal and are most often overlooked are errors occurring during post-PCR processing. Differences in sample pipetting and electroki- netic injection during CE can cause sample-to-sample vari- ability, making profile interpretation more difficult to assess. For other analytical applications involving CE, the technical processes can be monitored by use of an internal standard, i.e. a substance that is added to the target analyte and is added in even amounts to all samples including blanks, standards and exogenous controls [12]. As such, the internal standards generate a stable signal that can be used to normalise the analyte signal and reduce variability associated with injection volume, voltage and electrophoresis to give a more consistent result [13–15]. Normalisation of the analyte signal is typically done by dividing peak area of the analyte with that of the in- ternal standard to give the peak area ratio [16, 17]. Therefore, when a higher or lower injection volume is taken up, both the peak area of the analyte and that of the internal standard will increase or decrease proportionally, while the peak area ratio remains constant [17, 18]. With the application of CE to DNA profiling, the role of the internal standard is partly taken over by the internal size standard (ISS). This is added to the PCR product after ampli- fication and before CE and is used to size the amplified DNA fragments. In addition to this, because it is mixed and injected with the PCR products, it can be used to show the success of the injection. However, no data normalisation is done in ∗ Current Address: Dr. Zahra Nathalie, Department of Genetics, Univer- sity of Leicester, UK. Colour Online: See the article online to view Figs. 1 and 2 in colour. C  2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com