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Applied Biosystems VeriFiler Plus PCR Amplification Kit with internal quality control system provides confident answers with challenging forensic samples
The Applied Biosystems™ VeriFiler™ Plus PCR Amplification Kit has been developed to meet challenging forensic casework needs globally. It is a companion kit to the Applied Biosystems™ VeriFiler™ Express PCR Amplification Kit for databasing. Powered by a six-dye short tandem repeat (STR) chemistry and an improved master mix formulation, this kit is specifically designed for challenging sample types—touch, degraded, or inhibited samples with improved sensitivity and enhanced robustness against inhibition. With an recommended input of 0.5 ng, the VeriFiler Plus kit targets a total of 25 loci- 23 autosomal STRs, including the core CODIS loci, Penta D, Penta E and D6S1043, 2 gender discrimination markers, while also including an internal quality control (IQC) system. The IQC system contained in the VeriFiler Plus kit consists of two synthetic sequences with specific primers for each of the targets. This system provides positive confirmation that all assay components are functioning as expected. This system is particularly useful for confirming the validity of negative results and distinguishing samples that are degraded from those that contain PCR inhibitors.
Forensic casework samples may be collected from substrates that introduce a wide range of impurities that are inhibitory to PCR amplification, and/or could have DNA molecules that have been degraded by environmental insults. When performing casework sample analysis, it may be helpful to have a system that generates a positive signal that could confirm DNA-negative results, while distinguishing between samples that were challenged by PCR inhibitors, versus those that contain degraded DNA. Inhibited and degraded DNA samples can have a similar appearance in CE electropherograms but require different treatments to obtain optimal results. For these reasons, we developed the VeriFiler Plus Kit with an IQC system.
2. Materials and methods
2.1 Sensitivity test
Serial two-fold dilutions of the VeriFiler Plus kit male Control DNA 007 were made to yield a final input of 1 ng to 16 pg per reaction. The samples were tested in four internal and four external labs following the experimental conditions described below in Section 2.3.
2.2 IQC evaluation
The inhibited samples were created by adding humic acid (Sigma) to the control DNA 007 to a final concentration of 350 ng/ul. Degraded DNA was created by a combination of mechanical shearing and enzymatic degradation of 9947A DNA control (Marligen Biosciences). Inhibited, degraded, and control samples were analyzed using 500 pg input and the standard VeriFiler Plus kit protocol.
2.3 Experimental conditions
The VeriFiler Plus Kit has been optimized in a 25 uLtotal reaction volume, consisting of 2.5 μl of primer mix, 5 μl of master mix, and 17.5 μl of sample input volume. The thermocycling protocol is described in the VeriFiler Plus User Guide [
]. The allele peaks were interpreted using a 175 RFU peak amplitude threshold. The five dyes used in the VeriFiler Plus Kit to label amplified sample products are 6-FAM™, VIC™, TED™, TAZ™, and SID™ dye labels. The sixth dye, LIZ™, is used to label the GeneScan™ 600 LIZ Size Standard v2.0.
3. Results and discussion
3.1 Sensitivity test
The VeriFiler Plus kit was developed to process samples with low DNA concentration. It features a highly concentrated primer mix at 10X and master mix at 5 × . This allows up to 17.5 μl of sample input in a 25 μl reaction volume, and thus increases the chance of generating profiles from low-DNA samples. The Verifiler Plus kit also utilizes a brighter fluorescent dye TED to replace NED™ dye used in Globalfiler. This change boosts its signal – compared with the Globalfiler kit standard input amount of 1 ng DNA, the VeriFiler Plus kit achieved optimal peak height with 0.5 ng DNA in 29 PCR cycles. Internal and external tests in 8 laboratories showed that the VeriFiler Plus kit is able to recover a full profile reproducibly with 125 pg of DNA input (Fig. 1). Significant number of alleles can still be genotyped with DNA inputs of 16 pg.
Fig. 1VeriFiler Plus Kit allele recovery with different DNA inputs from 8 different test sites (n = 6 replicates). A complete profile consists of 46 alleles (shown in green boxes). Partial profiles are shown in yellow boxes. Failed capillary electrophoresis injections are shown as grey boxes with no numbers shown. The average number of alleles amplified at each input amount are shown on the left as a percent of the possible total. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
The IQC system is a tool that can help evaluate the PCR reaction and, along with the STR marker data, infer possible sample degradation or inhibition.The primers for the two IQC markers, IQCS (small) and IQCL (large), amplify synthetic DNA targets that are included in the primer mix. IQCS is a low molecular weight amplicon, with mobility of 70 nt. IQCL is a higher molecular weight amplicon, with mobility of 436 nt. The IQC system allows the confirmation of the success or failure of the PCR reaction, by the presence or absence of the IQCS and IQCL primer peaks on the electropherogram. The IQC system can also help determine if PCR inhibitors might be present in the PCR reaction, or if the PCR reaction conditions are not optimal, by evaluating the relative peak heights of IQCS and IQCL.
The peak heights of the IQCS and IQCL are indicators of PCR performance. Under ideal PCR conditions, the peak heights of the IQCS and IQCL should be >2000 RFU using standard injection protocols on the 3500/3500xL Genetic Analyzer. For a valid negative run, the user can confidently confirm that the negative result is due to no sample input instead of PCR failure (Fig. 2, top). The IQC system is also a powerful tool to distinguish samples compromised by inhibition and/or degradation. Both types of samples give similar ski-slope effect profiles, but give different relative IQC peak heights. The height of the IQCL remains high with the degraded samples (Fig. 2, middle), while the height of the IQCL is substantially reduced with the inhibited samples (Fig. 2, bottom). If the IQC system indicates degraded DNA, forensic analysts may reamplify the sample with a higher amount of input DNA or choose a STR amplification kit that has an alternative marker set configuration to maximize information recovery. If the IQC system indicates the presence of inhibitors, the analyst may opt for an additional purification step or a dilution of the original sample before repeating sample amplification.
Fig. 2IQC profiles and interpretation with different sample types.
The VeriFiler Plus kit with the IQC system is ideally suited for genotyping challenging casework samples. The IQC system can effectively differentiate inhibited from degraded samples, enabling qualitative insight into the amplification step while informing an optimal course of action for the sample.
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