Time and labor savings continue to be key goals for database and paternity labs. Increasing sample numbers and tightening government budgets necessitate that database laboratories seek process improvements. Similarly, efficiency gains are key for commercial paternity testing organizations to remain competitive.
Recently, the STR-typing community has begun to adopt the concept of “direct amplification” from unpurified samples on FTA® cards. Significant time and labor savings can be achieved by eliminating DNA purification and amplifying samples directly from buccal or blood samples on FTA® cards. Another time-consuming process is the PCR itself—often three hours or more. The duration of this step often dictates daily workflow for laboratories, and when combined with DNA purification and capillary electrophoresis, the process often requires more than one day. Although thermal cycling is a hands-off step, decreasing sample time on an instrument with rapid amplification allows samples to be processed in a single day.
The PowerPlex® 18D System allows direct amplification and reduced thermal cycling time to provide a simpler and faster genotyping system for database and paternity laboratories. Additionally, the 18 loci amplified represent all markers commonly typed in CODIS laboratories.
“Significant time and labor savings are achieved through robust direct amplification and rapid PCR.”
The PowerPlex® 18D System
The PowerPlex® 18D System (Cat.# DC1802 and DC1808) was developed for direct amplification of buccal or blood samples on FTA® cards. The five-color system (Figure 1) contains primers for amplification and genotyping of 17 STR loci (D2S1338, D19S433, Penta E, Penta D, D3S1358, TH01, D21S11, D18S51, D5S818, D13S317, D7S820, D16S539, CSF1PO, vWA, D8S1179, TPOX and FGA) as well as Amelogenin, encompassing all 13 CODIS loci. One primer for each locus is labeled with fluorescein (blue), JOE (green), TMR-ET (yellow) or CXR-ET (red). Detection is performed with the Applied Biosystems 3100, 3130 or 3500 Genetic Analyzer. Amplified samples can be sized with the supplied CC5-labeled Internal Lane Standard 500 (CC5 ILS 500). Proper color deconvolution is achieved by matrix standardization or spectral calibration using the PowerPlex® 5-Dye Matrix Standards (Cat.# DG4700). Finally, a new control DNA, 2800M, is provided in the kit (Figure 2). This DNA is derived from a single-source, human blood sample.
Figure 1. Configuration of the PowerPlex® 18D System.
The PowerPlex® 18D System contains all 13 CODIS loci: D3S1358, THO1, D21S11, D18S51, D5S818, D13S317, D7S820, D16S539, CSF1PO, vWA, D8S1179, TPOX and FGA, plus Amelogenin, Penta E, Penta D, D19S433 and D2S1338.
Figure 2. Amplification of the 2800M Control DNA.
Five nanograms of 2800M Control DNA was amplified for 27 cycles on a GeneAmp® PCR System 9700 using the PowerPlex® 18D System. Samples were analyzed using a 3kV, 5-second injection and a 1,500-second run time on an Applied Biosystems 3130xl Genetic Analyzer.
Direct Amplification with the PowerPlex® 18D System
While the PowerPlex® 18D System can be used to amplify extracted samples, such as purified reference samples, the kit was optimized for direct amplification from samples on FTA® cards. Components in FTA® cards lyse cells contained in the deposited sample and promote DNA accessibility for PCR. However, components that drive lysis can inhibit PCR; thus, washing or purification protocols are generally incorporated prior to amplification to type samples on FTA® cards successfully. This is not necessary with PowerPlex® 18D. Core punches are taken from sample-spotted FTA® cards and placed directly into the PowerPlex® 18D reaction. The master mix in the PowerPlex® 18D System overcomes inhibitors without washing or purification (Figures 3 and 4).
Figure 3. Amplification of buccal samples on FTA® cards.
A buccal sample was collected and immediately transferred to FTA® paper. Reactions were set up using one (Panel A) or two (Panel B) 1.2mm punches from the same card. Amplifications were performed using 27 cycles on a GeneAmp® PCR system 9700. One microliter of amplified sample was analyzed using a 3kV, 3-second injection and 1,500-second run time with the G5 dye set on an Applied Biosystems 3130xl Genetic Analyzer.
Figure 4. Amplification of blood samples on FTA® cards.
Whole blood (125µl of K2 EDTA-anti-coagulated blood) was spotted onto FTA® paper. Reactions were set up using one 1.2mm punch and amplified using 27 cycles on a GeneAmp® PCR system 9700. One microliter of amplified sample was run with a 3kV, 3-second injection and 1,500-second run time with the G5 dye set on the Applied Biosystems 3130xl Genetic Analyzer.
With direct amplification, sampling can affect results more than with standard amplifications. Although a typical 1.2mm punch contains sufficient material for successful amplification, the distribution of sample across an FTA® card varies, most notably with buccal samples. Indicating FTA® cards help locate a buccal sample on the card and, therefore, the ideal area to punch. However, indicating cards only show where the surface was wetted. Wetted but bare areas of the card could exist, and sampling from those areas will not result in amplification. Failures due to bare punches can be resolved by including two punches per reaction, increasing the probability that the reaction contains at least one sample-covered punch and increasing first-pass success rates. As shown in Figure 5, only a slight increase in peak height may be seen for reactions with two punches compared to those with one punch. However, the PowerPlex® 18D System can accommodate two buccal punches. For blood samples on FTA® cards, one punch is recommended. The use of two blood punches is not necessary, as sample distribution is clearly visible and generally more consistent than buccal punches (Figure 5). In many cases, the use of two FTA® punches when working with blood on FTA® cards can actually result in poorer results due to an increase in PCR inhibitors associated with blood samples on FTA® paper.
Figure 5. Signal from one or two FTA® punches.
FTA® cards from two buccal donors (Panel A) and two blood donors (Panel B) were amplified using one or two punches per reaction, with three replicates each. The average locus peak height of three replicates for each condition is shown.
Previous commercial STR kits were optimized for older thermal cycling technology and performance of the enzyme available at the time. Cycle times generally approach or exceed 3 hours (Figure 6). Consequently, processing each plate of samples from sample to analysis, including extraction, cycling and capillary electrophoresis (CE) analysis, requires more than one day. Thermal cycling time for the PowerPlex® 18D System is less than an hour and a half using a simple, two-step cycling program (Figure 6). With direct amplification, samples that are punched in the morning are immediately ready for PCR. Less than 2 hours later, those same samples are ready to be placed on the CE instrument. This simplified process reduces total hands-on time as well as the number of manipulations required, saving staff resources and reducing the chance of error.
Figure 6. Thermal cycling programs.
The approximately 3 hour, 3-step cycling program for PowerPlex® 16 HS System is shown (left) and compared to the approximately 80-minute, two-step cycling program for PowerPlex® 18D System (right).
The PowerPlex® 18D System was developed to simplify and accelerate STR typing for database and paternity testing. With 18 loci the system is compatible with existing databases, allowing maximum data sharing between CODIS laboratories and future results to build on those databases. In addition to significant time and labor savings through robust direct amplification and rapid PCR, the PowerPlex® 18D System has the flexibility and performance to accommodate the sample types currently submitted to laboratories and successfully meet laboratory goals. Consequently, the PowerPlex® 18D System is an effective option to increase lab efficiency and simplify sample process workflow.