|Year : 2015 | Volume
| Issue : 1 | Page : 3-7
Evaluation of the AGCU Expressmarker 16 and 22 PCR Amplification Kits Using Biological Samples Applied to FTA Micro Cards in Reduced Volume Direct PCR Amplification Reactions
Samantha J Ogden, Kathryn L Lamerton, Peter J Tatnell
R&D, Bio-Research Tools, GE Healthcare Life Sciences, The Maynard Centre, Forest Farm, Whitchurch, Cardiff, United Kingdom
|Date of Web Publication||29-May-2015|
Kathryn L Lamerton
GE Healthcare Life Sciences, The Maynard Centre, Forest Farm, Whitchurch, Cardiff CF14 7YT
Source of Support: None, Conflict of Interest: None
This study evaluated the performance of the Wuxi AGCU ScienTech Incorporation (HuiShan, Wuxi, China) AGCU Expressmarker 16 (EX 16) and 22 (EX22) short tandem repeat (STR) amplification kits in reduced reaction volumes using direct polymerase chain reaction (PCR) amplification workflows. The commercially available PowerPlex® 21 (PP21) System (Promega, Wisconsin, USA), which follows similar direct workflows, was used as a reference. Anticoagulate blood applied to chemically impregnated FTA TM Micro Cards (GE Healthcare UK Limited, Amersham Place, Little Chalfont, Buckinghamshire, HP7 9NA, UK) was used to represent a complex biological sample. Allelic concordance, first-pass success rate, average peak heights, heterozygous peak height ratios (HPHRs), and intracolor and intercolor peak height balance were determined. In reduced volume PCR reactions, the performances of both the EX16 and EX22 STR amplification kits were comparable to that of the PP21 System. The level of performance was maintained at PCR reaction volumes, which are 40% of that recommended. The EX22 and PP21 System kits possess comparable overlapping genome coverage. This study evaluated the performance of the AGCU EX16 and EX22 STR amplification kits in reduced PCR reaction volumes using direct workflows in combination with whole blood applied to FTA TM Micro Cards. Allelic concordance, first-pass success rate, average peak heights, HPHRs, and intracolor and intercolor peak height balance were determined. A concordance analysis was completed that compared the performance of the EX16 and EX22 kits using human blood applied to FTA Micro Cards in combination with full, half, and reduced PCR reaction volumes. The PP21 System (Promega) was used as a reference kit. Where appropriate, the distributions of data were assessed using the Shapiro-Wilk test. For normally-distributed data, statistics were calculated using analysis of variance (ANOVA) and for nonparametric data the Wilcoxon/Kruskal-Wallis test was used. Statistical significance was set at P < 0.05. Confidence intervals for mean values were set at 95%. On using reduced volume PCR reactions in combination with dried blood spots applied to FTA sample collection cards, both the EX16 and EX22 kits were shown to generate STR profiles of sufficient quality to allow entry into National DNA databases. The performance of both EX16 and EX22 was comparable to that of the PP21 System. This study demonstrates the successful use of the Wuxi AGCU ScienTech Incorporation EX16 and EX22 kits in reduced PCR reaction volumes with complex biological samples applied to chemically impregnated FTA sample collection cards.
Keywords: Direct amplification, DNA typing, dried blood spots, expressmarker (EX), forensic DNA analysis, forensic science, FTA sample collection cards, short tandem repeat (STR) profiling
|How to cite this article:|
Ogden SJ, Lamerton KL, Tatnell PJ. Evaluation of the AGCU Expressmarker 16 and 22 PCR Amplification Kits Using Biological Samples Applied to FTA Micro Cards in Reduced Volume Direct PCR Amplification Reactions. J Forensic Sci Med 2015;1:3-7
|How to cite this URL:|
Ogden SJ, Lamerton KL, Tatnell PJ. Evaluation of the AGCU Expressmarker 16 and 22 PCR Amplification Kits Using Biological Samples Applied to FTA Micro Cards in Reduced Volume Direct PCR Amplification Reactions. J Forensic Sci Med [serial online] 2015 [cited 2019 Jan 22];1:3-7. Available from: http://www.jfsmonline.com/text.asp?2015/1/1/3/155546
| Introduction|| |
Chemically impregnated sample collection cards, such as FTA TM (GE Healthcare, GE Healthcare UK Limited, Amersham Place, Little Chalfont, Buckinghamshire, HP7 9NA, UK) are used for generating short tandem repeat (STR) profiles during forensic investigations. , FTA cards contain chemicals that are capable of inhibiting STR reactions, therefore in forensic workflows, any inhibitory effect derived from either the FTA card or the crude biological sample needs to be evaluated. , Recently, the AGCU Expressmarker 16 (EX16) and 22 (EX22) STR loci Direct PCR Amplification kits (Wuxi AGCU Scien Tech Incorporation: No.18-1,WenHui Rd.,HuiShan District,Wuxi,China) demonstrated successful STR amplification from blood and buccal samples applied to FTA cards without the need for DNA purification. , Reducing polymerase chain reaction (PCR) volumes offers an opportunity for using lower amounts of DNA and reducing costs.
| Materials and Methods|| |
All experiments were performed according to the guidelines described in the Scientific Working Group on DNA Analysis Methods. 
FTA Micro Cards; sample application and processing
This investigation was designed to evaluate the EX16 and EX22 kits in a STR-based concordance study using reduced volume PCR reactions with human blood applied to FTA Micro Cards. The PowerPlex® 21 (PP21) System (Promega, Wisconsin, USA) was used as a reference; this system has been optimized for many sample types, including the direct PCR amplification of DNA from samples applied to FTA Micro Cards. Anticoagulated blood (75 μL aliquots) was spotted onto FTA Micro Cards and allowed to dry at ambient temperature for 3 h. For PCR-based STR amplifications, three 1.2 mm-diameter punches were excised from a single FTA dried blood spot and each punch was placed into separate reaction mixtures. In total, punches were excised from 30 dried blood spots. The FTA punches were not subjected to a washing step prior to use but were added directly to STR reaction mixtures in accordance with direct amplification workflows.
DNA amplification and electrophoresis
For the EX16 and EX22 kits, PCR reactions were generated according to the manufacturer's instructions and contained a 1.2 mm punch excised from an FTA dried blood spot. Full (25 μL), half (12.5 μL), and low (10 μL) PCR reaction volumes were used. STR products were amplified in a MicroAmp Optical 96-well reaction plate ( Life Technologies, Life Technologies Ltd, 3 Fountain Drive, Inchinnan Business Park, Paisley PA4 9RF, UK) using a GeneAmp PCR system 9700 (Life Technologies). The EX16 thermal cycle consisted of 94°C for 1 min, followed by 32 cycles of 98°C for 5 s, 59°C for 15 s, and 72°C for 10 s. A final extension step of 72°C for 5 min was performed. The EX22 thermal cycle consisted of 95°C for 2 min, followed by 10 cycles of 94°C for 30 s, 60°C for 1 min, and 72°C for 1 min; and 20 cycles of 90°C for 30 sec, 58°C for 1 min, and 72°C for 1 min; with a final step of 72°C for 10 min. Reaction mixtures and thermal cycling parameters for the PP21 System were according to the manufacturer's instructions. Amplification products were analyzed using an Applied Biosystems (ABI, 3 Fountain Drive, Inchinnan Business Park, Paisley PA4 9RF, UK) 3130XL Genetic Analyzer capillary electrophoresis system. For products amplified using the EX16 and EX22 kits, the run cocktail mix was prepared by adding AGCU Marker SIZ-500 ( Wuxi ScienTech, Wuxi AGCU Scien Tech Incorporation: No.18-1,WenHui Rd.,HuiShan District,Wuxi,China) reagent and Hi-Di Formamide to the PCR products. Instrument settings were: Injection time 10 s; injection and run voltages 3 kV; run time 2,500 s. For samples amplified using the PP21 System, the run cocktail mix was prepared by adding the CC5 internal lane standard 500 (Promega) and Hi-Di Formamide to the PCR products. Instrument settings were as described above, except that injection time and run time were reduced to 5 s and 1,800 s respectively. Results were evaluated using the GeneMapper™ ID v3.2 software (Life Technologies).
Reduced volume PCR reactions
The number of STR profiles that exhibited full, partial, or no profiles, the average peak heights, and the numbers of alleles present in each sample were determined. Full profiles were categorized as alleles present at each loci with a peak height of greater than 75 relative fluorescence units (RFU) for heterozygote peaks and 150 RFU for homozygote peaks. All loci with heterozygote peaks must exhibit a peak height ratio greater than 50%. Partial profiles were defined by missing allelic peaks, the presence of "off-ladder peaks," and loci with heterozygous peaks with an intralocus balance less than 50%. "No profiles" were defined as the absence of any peak > 50 RFU. First-pass success rates were defined as the successful generation of a full STR profile on the first attempt without subsequent reprocessing. These RFU values are based on those stipulated by the custodian of the UK National DNA database. 
Peak height ratios were calculated by dividing the lower allele peak height of a heterozygous individual by the higher allele peak height at a given locus and the result expressed as a percentage. Intracolor peak balance was calculated by first averaging heterozygous peak heights and dividing homozygous peak heights by 2. Once normalized for diploidy, the lowest score for a locus labeled with a given dye was divided by the highest and the result reported as a percentage. Intercolor balance was calculated by dividing the lowest normalized score for a locus within a sample by the highest score and the result expressed as a percentage (regardless of dye color). These calculations are based on those described by Wang et al., 2011. 
| Results|| |
On using FTA Micro Cards spotted with blood, the PP21 System EX16 and EX22 kits produced concordant alleles for all shared loci [Table 1]. All the kits possess similar overlapping genome coverage, and amplified the 21, 16, and 22 loci respectively. Amplification of the D1S1656 locus was only possible using the PP21 kit, while amplification of the D2S441 and D10S1248 loci was only possible using the EX22 kit.
|Table 1: Concordance of STR Profiles Derived from Blood Applied to FTA Micro Cards and Genotyped Using the EX16 Kit, the EX22 Kit, and the PP21 System |
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Reduced volume PCR reactions
When single 1.2 mm-diameter FTA dried blood spots were used in 25 μL (full) PCR volumes, the EX16 kit generated full STR profiles with a 100% first-time pass success rate, while the PP21 System and EX22 success rates were 90% and 53% respectively [Table 2]. For the EX22 kit, 14 of the 30 (47%) STR profiles at the penta E loci produced heterozygous peak height ratios (HPHRs) of less than 50% and were therefore defined as partial profiles, resulting in a lower first-time success rate. The average peak heights for these 14 penta E alleles were all > 2,000 RFUs and were therefore of sufficient quality for acceptance into the UK National DNA database. Peak height imbalance can be caused by low amounts of template DNA, degradation of the sample, PCR inhibition, and preferential amplification.  Considering the sample type used for this study, the peak height imbalance observed at the penta E is probably due to preferential amplification. This is a common occurrence for loci associated with larger PCR products and, in particular, where one heterozygous allele is significantly longer than the other. In this study, the penta E allelic lengths were 7 and 17 respectively, and the larger fragment may have been amplified less efficiently.
|Table 2: Number of Full, Partial, and No Profiles Obtained from Dried Blood Spots on FTA Micro Cards using different PCR Volumes |
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Three (out of 30) partial profiles obtained with the PP21 System were due to off-ladder peaks. These peaks were located outside the specific sizing range of the alleles associated with the D12S391 locus and were caused by "pull-up" in the profile.
For half-volume (12.5 μL) reactions, the PP21 System and the EX16 and EX22 kits generated first-time pass rates of 93%, 100%, and 100% respectively. The EX16 kit produced two partial profiles due to the failure of size standards and poor peak morphology. If repeated, it is considered likely that these samples would produce full profiles.
When used in low volume (10 μL) PCR reactions, all three kits generated first-pass success rates of 100%.
Irrespective of the PCR reaction volume used, all three STR amplification kits generated profiles that were of sufficient quality to allow uploading to the UK National DNA database. 
|Table 3: Average Peak Heights Obtained from Dried Blood Spots on FTA Micro Cards Using Different PCR Volumes |
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Average peak heights
The average peak heights generated from PCR amplifications performed using FTA dried blood spots and reduced PCR volumes are shown in [Table 3]. When used in 25 μL (full) PCR volumes, all three kits generated statistically similar average peak heights (P > 0.05). However, when used in 12.5 μL (half) and 10 μL (low) PCR volumes, the EX22 kit produced significantly higher average peak heights compared to both the EX16 kit and the PP21 System (P < 0.05). A similar increase in average peak heights was observed using the AmpFLSTR® Profiler Plus® kit (AmFLSTR® Profiler Plus® kit: Applied Biosystems: 3 Fountain Drive, Inchinnan Business Park, Paisley PA4 9RF, UK) at reduced volumes,  and this suggests that DNA is amplified more efficiently, generating more PCR products, when reactions are performed in reduced volumes. Reduced volume PCR reactions can, therefore, provide improved conditions that permit more efficient PCR amplification conditions, resulting in the subsequent generation of higher peak heights.
The HPHR obtained at each PCR volume [Table 4] indicates that the pair of alleles associated with each heterozygous locus was relatively balanced, irrespective of the kit or reaction volume used. The PP21 System produced higher average HPHR values (ranging 87.5-92.5%; mean 90.2% ±2.5) compared to the EX16 and EX22 kits (average HPHR ranged 79.6-85.2%; mean 81.9 ± 2.9 and 80.4%-83.2%; mean 81.4 ± 1.6 respectively). HPHRs associated with the EX22 kit tended to exhibit the lowest minimum value and the greatest spread between the minimum and maximum values. For example, the EX22 HPHR values associated with 25 μL reaction volumes ranged 50.4-98.4%. For both the EX16 kit and the PP21 System, a trend of small, incrementally decreasing median HPHR values with reducing PCR volumes was observed. However, for the EX22 kit, the median HPHR values remained relatively constant irrespective of reaction volume.
|Table 4: Average Heterozygous Peak Height Ratios Obtained from Dried Blood Spots Applied to FTA Micro Cards Using Different PCR Volumes |
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Intra - and intercolor peak balance
Using the FTA dried blood spots, the mean intracolor peak height balance in each fluorescent dye channel was calculated for all three kits at each PCR volume. All kits irrespective of the PCR volume generated good quality STR profiles, producing well-balanced peak heights within each dye channel. The PP21 System and the EX16 and EX22 kits produced intracolor peak height balances ranging 51.6-83.8%, 48.9-79.0%, and 55.4-66.8%, respectively (data not shown).
The intracolor peak balance was calculated to assess the overall color balance within specific dye lanes at reduced PCR volumes. This is important when assessing the condition of the sample. The intracolor peak height ratios determined for both the EX16 and EX22 kits are comparable to those described during the amplification of nondegraded and uninhibited samples of human DNA using the AmpFLSTR Identifiler PCR Amplification Kit in which the average intracolor balance was calculated as > 50%. 
These data were also analyzed to generate intercolor peak height balance across all four dye channels and PCR volumes. The EX22 kit produced the greatest intercolor peak heights across the four dye channels, the values ranging from 48.1% (low volume) to 58.9% (full volume). Values derived from the EX16 and PP21 System kits were comparable but lower, ranging 16.3-41.6% and 13.3-33.6% respectively. For all three kits, the percentage intercolor peak height balance decreased with reducing PCR volume. The intercolor balance determined during the validation of the AmpFlSTR Identifiler PCR Amplification Kit was reported to be 20-40%.  This range is comparable to those determined for both the EX16 and PP21 System kits, but lower than that observed using the EX22 kit.
ES cells on FTA cards
The performance of both EX kits was also evaluated using human embryonic stem (hES) cells applied to FTA Micro Cards (data not shown). hES cells were diluted to approximately 20,000 cells in 50 μL phosphate-buffered saline and applied to FTA Micro Cards. After drying, PCR-based STR amplification reactions were performed using a single 1.2 mm FTA punch (in replicates of five), and analyses similar to those described earlier were performed. Essentially, results using hES cells as the biological sample were comparable to those when using dried blood spots, and the performance of both EX kits was similar to that of the PP21 System.
| Discussion|| |
This study assessed the performance of the EX16 and EX22 STR profiling kits in reduced PCR volumes when used with biological samples applied to FTA sample collection cards. The resultant data indicated that both EX kits generated good quality STR profiles in direct amplification workflows that were comparable to those generated using the Promega PP21 System.
When using full (25 μL), half (12.5 μL), and low (10 μL) PCR volumes, the EX16 and EX22 kits and the PP21 System kits produced 100% concordant alleles at all loci when STR markers were amplified directly from blood applied to FTA Micro Cards.
Investigation into first pass success rate for generating full STR profiles
In terms of generating full STR profiles at the first attempt, the performance of all three kits was comparable when used in combination with reduced PCR volumes; however, EX22 performed less well with full PCR reaction volumes. This may be related to the presence of two alleles at the heterozygous penta E loci that were significantly different in length. The EX22 kit gave marginally superior performance when used in reduced PCR reaction volumes compared to both the EX16 kit and the PP21 System, producing higher average peak heights, more consistently balanced HPHRs, comparable intracolor peak height balance, and higher intercolor peak height balance.
The observed marginally better performance of the EX22 kit when using dried blood spots as the biological sample applied to FTA cards may be related to several factors. The EX22 reaction components may exhibit increased resistance to potential inhibitors in the blood compared to the formulation present in the EX16 kit. Heme is known to be a potent inhibitor of PCR-based nucleic acid amplification reactions.  An additional factor could be related to the known variation in the distribution of biological samples across cellulose-based solid support materials. Direct PCR amplification kits use a small portion from a heterogeneous, dispersed sample with localized areas of concentration, and as a result of this distribution, the location of sampling on a solid support material contributes significantly to STR parameters such as peak height variation. , These factors, together with the assumption that DNA in a smaller PCR volume could be more available for binding by primer or polymerase molecules because the overall amount of DNA is less dilute than in a higher volume,  could explain the marginally better performance of the EX22 kit when used in combination with FTA dried blood spots.
The successful generation of STR profiles and other DNA typing methods from biological samples depends on the optimization of numerous factors, e.g. reducing the effect of nucleic acid amplification inhibitors. An additional important and critical factor that ensures reliability is the use of the most appropriate volume for delivering an efficient amplification process,  and this study demonstrates that both AGCU EX16 and EX22 STR amplification kits when used in reduced volume PCR reactions can generate STR profiles of sufficient quality for submission to National DNA databases. This performance was delivered using whole blood as a representative, complex biological sample applied to chemically impregnated FTA sample collection cards. Blood and the chemistry associated with FTA sample collection cards are known to contain potent nucleic acid amplification inhibitors. The level of performance of both EX kits is maintained when using PCR reaction volumes that are 40% lower than the volume recommended by the manufacturer and is comparable to the PP21 System.
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[Table 1], [Table 2], [Table 3], [Table 4]