Use PowerPlex_R_16 HS system to directly amplify blood card and oral sample card

Introduction

Short tandem repeat (STR) analysis is currently a common technique in forensic science and paternity testing laboratories. For known samples, such as paternity tests, detection of victims or suspects, oral and blood samples are often used. Such specimens are often collected on special cards, such as FTA® and Protein Saver® 903 cards. These cards contain special chemical reagents that can lyse cells and protect DNA for subsequent detection and analysis. These cards can be stored at room temperature for a long time, so it provides a simple, stable, and cheap way to store DNA samples.

Normally, blood and oral samples contain substances that inhibit DNA amplification. Previously, we usually extracted DNA from these paper cards and purified it to remove inhibitors. However, if we can directly amplify the protected DNA in the paper, it will allow us to analyze DNA samples faster and more efficiently. By omitting DNA purification steps, users can save reagent costs and shorten test time. Forensic science database building laboratories and paternity testing laboratories usually have a large number of samples to analyze, so direct amplification provides an efficient and stable method for these laboratories.

Promega recently introduced the PowerPlex® 16 HS system (Cat. # DC2101), which can co-amplify 16 STR sites. The PowerPlex® 16 HS system incorporates hot-start Taq DNA polymerase, and compared with other STR products, including the original PowerPlex® 16 system, the HS system is more resistant to PCR inhibitors, which makes PP16 HS very Suitable for direct amplification. Here, I demonstrated the effectiveness of using the PowerPlex® 16 HS system to directly amplify oral and blood samples on FTA® and Protein Saver® 903 cards.

Materials and Methods

The oral samples of 11 volunteers were collected using the EasiCollect ™ sample collection card (Whatman, Kent, UK) according to the procedures in the operation manual. Eleven blood samples were collected using Vacutainer® EDTA anticoagulated blood collection tubes (Becton Dickinson, Franklin Lakes, NJ), and then made into FTA® cards and Protein Saver® 903 cards. All samples are thoroughly dried before extraction.
We tested cards with a punching diameter of 0.5-2.0 mm, and finally selected a 1.2 mm piece of paper. We punched four 1.2 mm paper sheets from each card, and placed each one in a 96-well amplification plate. The PowerPlex® 16 HS system was used to directly amplify the STR sequence of the sample. The 25ul reaction system contains 5μl 5X Master Mix, 2.5μl 10X Primer Pair Mix and 17.5μl Water, Amplification Grade, these components are amplified together with the paper.

The amplification is performed on the GeneAmp® PCR System 9700 thermal cycler. For the amplification procedure, refer to the PowerPlex® 16 HS system technical manual (# TMD022, # CTMD022). We tested 26-32 cycles, and finally selected 28 cycles (10/18) for amplification. 1 μl of the amplified product was used for electrophoretic detection on an ABI PRISM® 3100 Genetic Analyzer (Applied Biosystems) using a 3 kV voltage and 5-second injection. Electrophoresis data was analyzed using GeneMapper® ID software version 3.2. Refer to the PowerPlex® 16 HS system technical manual (# TMD022, # CTMD022) for analysis settings.

Result analysis

An important feature of direct amplification is the ability to obtain stable and repeatable genetic map results.

EasiCollect â„¢ oral sample card

Of the 44 oral sample results, 43 obtained a complete genetic map, and only one sample did not complete the map, and 6 of the 31 alleles were lost. In other words, among the 4 cards taken, 3 of them got the complete map. In summary, the success rate of oral samples in obtaining complete maps is 98%. The peak heights of heterozygotes in the spectrum of 44 samples ranged from 2709 ± 1659 RFU (Table 1). Most of the peak heights of heterozygotes were between 1000–4000 RFU, and the average peak height of the same human source sample was stable. In addition, the balance between the sites also performed well. Figure 1, Panel A shows a typical representative EasiCollect ™ oral sample.

FTA® card-blood sample

Of the 44 FTA® card blood sample results, 43 obtained a complete genetic map. Another sample could not be analyzed due to internal standard (ILS600) electrophoresis failure. Therefore, all the 43 analyzable samples obtained a complete genetic map (100%). The peak height of heterozygotes in the spectrum of 43 samples was 1736 ± 1009 RFU (Table 1), and most of the peak heights of heterozygotes were in the range of 1000–2500 RFU. The average peak height of the same human source sample is stable. Figure 1, Panel B shows a representative FTA® card blood sample.

903 Card-Blood Sample

Of the results of 44 903 card blood samples, 43 had a complete genetic map. The other sample could not be analyzed due to poor capillary injection and electrophoresis failure. Therefore, all the 43 analyzable samples obtained a complete genetic map (100%). The peak height of heterozygotes in the spectrum of 43 samples was 862 ± 516 RFU (Table 1), and most of the peak heights of heterozygotes were between 500–1500 RFU. The average peak height of the same human source sample is stable. Figure 1, Panel C shows a representative 903 card blood sample.

Figure 1. Electrophoresis results of three oral samples of different human samples after direct amplification

Matters needing attention for successful direct amplification

Due to the presence of inhibitors, the use of DNA preservation cards for direct amplification in the past often resulted in the loss of alleles, but in terms of STR analysis, the need for complete genetic maps obtained by each laboratory is increasing. The recently introduced PowerPlex® 16 HS system can greatly meet this demand. For most samples, including case samples containing a large amount of inhibitory reaction, this system can provide a complete genetic map to the greatest extent possible. The tolerance of the PowerPlex® 16 HS system to PCR inhibitors is much higher than other similar products, so it can be obtained for the samples that failed to be amplified before (1–3). These qualities make PP16 HS an effective direct amplification STR system. In addition, users only need one kind of kit to complete the inspection of case samples and samples suitable for direct amplification.

For successful direct amplification, many parameter settings require special attention, including the type of sample, the type of acquisition card, the diameter of the punch, the number of amplification cycles, and the injection status of capillary electrophoresis. We initially screened many types of blood cards and oral sample cards, with diameters ranging from 0.5 mm to 2.0 mm, and amplification cycles from 26 to 32 cycles. There is a great correlation between the card punching diameter and the number of cycles, so the user needs to adjust the parameter settings according to the sample and card type when conducting this test. In our experiment, we used a 1.2mm diameter card and 28 cycles. The results show that these parameters are applicable to most cards and sample types. In addition, we tested the two smallest card diameters, 0.5mm and 0.75mm. Although reducing the punching diameter can reduce the amount of cards, static electricity at this time will greatly affect the experiment process. Moreover, the diameter of 1.2mm is very suitable for mechanical drilling, for example using the BSD automatic punching machine (Australia), so that the entire process can be integrated into automation.

Although we can see from our results that the method in the test is suitable for most cards and sample types, we still recommend that each laboratory optimize its own internal parameters, for example, slightly increase or decrease the number of cycles or Hole diameter. In addition, due to the different settings of the sensitivity and signal strength parameters of each laboratory instrument, the injection parameters should also be optimized. In the initial experimental results of many cases, the average peak height of the spectrum obtained by different types of cards and the balance between the sites are different, so further optimization within a certain range is needed to improve the results. In addition, some other collection cards and collection techniques have internal differences in the sample deposition process, which will affect the subsequent STR analysis.

in conclusion

In summary, we demonstrated the feasibility and effectiveness of using the PowerPlex® 16 HS system to directly amplify the DNA storage cards of the mouth and blood and perform STR analysis. The main advantage of this method is that it saves time and costs. Especially for databases and paternity testing laboratories, direct amplification can solve the large number of unchecked samples accumulated there, while alleviating the problem of tight funding. At the same time, this study also demonstrated that such a kit commonly used in case analysis can also be effectively applied to the direct expansion of databases and paternity identification. This will help the consistency of laboratory products and the consistency of results.

references

1. Ensenberger, MG and Fulmer, PM (2009) The PowerPlex® 16 HS System. Profiles in DNA 12 (1), 9–11.
2. McLaren, R. (2007) PowerPlex® 16 versus Identifiler® Systems—Sensitivity and effects of inhibitors, # AN156, Promega Corporation.
3. AmpFlSTR® MiniFiler ™ PCR Amplification Kit User Guide (2007), Applied Biosystems