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Different Effects of PCR Inhibitors on Multiplex STR Assays Asilomar Conference Dennis Y. Wang, Ph.D.*, Julio J. Mulero, Ph.D., and Lori K. Hennessy, Ph.D. Human Identification Group, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, CA 94404 ABSTRACT Figure 2. Low pH can affect PCR amplification The effects of six common PCR inhibitors on the AmpFℓSTR® Identifiler® and AmpFℓSTR® MiniFilerTM PCR Amplification Kits were investigated. For each PCR amplification kit, the impact on changes in buffer acidity was first thoroughly evaluated, followed by determining the threshold inhibitory concentration (concentration where allele dropout was first observed) for each PCR inhibitor. In all studies, it was found that AmpFℓSTR® MiniFilerTM PCR Amplification Kit consistently outperformed AmpFℓSTR® Identifiler® PCR Amplification Kit in the presence of PCR inhibitors. Experiments were also conducted to identify methods for improving the quality of STR profiles under inhibited PCR conditions. 1.75 mM Ca 50 ng/ul Collagen = 0.0035N = 0.0045N Figure 5. In AmpFℓSTR® MiniFilerTM, low pH caused incomplete +A addition, resulting in split-peak morphologies in FGA, D2, D7, and CSF CSF D2 D7 FGA No acetic acid added 0.003N acetic acid INTRODUCTION In forensic human identification, DNA samples recovered from crime scenes are often commingled with PCR inhibitors that present a great challenge for PCR amplification. Crime scene evidence such as blood and semen which are often found on soil, sand, wood or leaf litter can contain substances which co-extract with the perpetrator’s DNA and prevent PCR amplification. Textile dyes, leather and wood from interior crime scenes can also contain inhibitors that interfere with the DNA polymerase’s activity. The impact of these contaminants on the multiplex STR assays can vary from attenuation to complete inhibition of the amplification process, resulting in partial STR profiles or profiles with unusual peak morphology. In the present study, a systematic approach was utilized to evaluate the effect of six PCR inhibitors commonly found in forensic samples on two different multiplex PCR assays. Each multiplex PCR assay has unique primer sequences and PCR buffer formulation. The six common PCR inhibitors used in this study were: ● Hematin1 - the main oxygen carrier in blood. ● Indigo2 - the most common dye from denim. ● Melanin3 - a pigment present in skin and hair. ● Humic acid4 - the polyphenolic compounds found in soil and water samples. ● Collagen5 - a connective protein making up 90% of the organic fraction of bones. ● Calcium6 - another major component of bone samples. The effects of these inhibitors were evaluated with the AmpFℓSTR® Identifiler® and AmpFℓSTR® MiniFilerTM PCR Amplification Kits. For each multiplex PCR reaction, a range of inhibitor concentrations was included during PCR amplification. The amplification results were evaluated based on: 1) the ability of each multiplex PCR assay to generate full STR profiles, and 2) the quality of the STR profiles obtained. The results clearly illustrated an increase in tolerance to PCR inhibitors in the AmpFℓSTR® MiniFilerTM PCR Amplification Kit, attributable to the robustness of the new PCR buffer formulation, optimized PCR primer sequences and thermal cycling parameters. Furthermore, our investigation revealed that the split peak tendency for certain loci in AmpFℓSTR® MiniFilerTM PCR Amplification Kit under acidic PCR condition can be corrected by increasing the length of the final PCR extension step. The performances of AmpFℓSTR® Identifiler® and AmpFℓSTR® MiniFilerTM in the presence of increasing concentration of HCl or acetic acid were investigated. With AmpFℓSTR® Identifiler®, PCR inhibition was observed when HCl and acetic acid is greater than 0.0035N and 0.0045N, respectively. With AmpFℓSTR® MiniFilerTM, PCR inhibition was observed when HCl is greater than 0.0040N and full STR profiles was generated even in the presence of 0.0055N acetic acid. The highest concentration of the calcium and collagen used in this study are indicated on the respective graphs. Figure 3. Effect of each PCR inhibitors on AmpFℓSTR® Identifiler® and AmpFℓSTR® MiniFilerTM 0.004N acetic acid 0.005N acetic acid Split-peak morphology in four STR loci was observed in AmpFℓSTR® MiniFilerTM under low pH PCR condition. The degree of the split-peak morphology worsens with increasing concentration of acetic acid. The remaining five STR loci were not affected. Figure 6. Incomplete +A addition in AmpFℓSTR® MiniFilerTM under acidic condition can be corrected by increasing the final PCR extension time 45 min 60 min 75 min 90 min MATERIALS AND METHODS PCR Inhibitor Preparation7 Stock solutions of 100 mM hematin and calcium were prepared in 0.1N NaOH and 0.1N HCl, respectively. A 100 mM stock solution of indigo was prepared by dissolving the indigo powder in 0.2% Triton X-100 in ddH2O. Stock solutions of 1 mg/ml melanin and collagen were prepared in 0.5N NaOH and 0.1N acetic acid, respectively. Lastly, 1 mg/ml of humic acid was prepared in ddH2O. Dilution of stock inhibitor solutions were made using ddH2O. PCR Amplification, Data Collection and Data Analysis PCR amplification was performed according to the protocols provided by the manufacturer. The DNA input, PCR cycling condition and number of PCR cycles are shown in the table below. The performances of AmpFℓSTR® Identifiler® and AmpFℓSTR® MiniFilerTM in the presence of increasing concentration of PCR inhibitors were investigated. With all six PCR inhibitors tested, AmpFℓSTR® MiniFilerTM consistently outperformed AmpFℓSTR® Identifiler® in overcoming PCR inhibition. The threshold inhibitory concentration for each PCR inhibitor is listed in Table 1. Table 1. Threshold inhibitory concentration of each PCR inhibitor for AmpFℓSTR® Identifiler® and AmpFℓSTR® MiniFilerTM Threshold Inhibitory Concentration = the lowest concentration of inhibitor that caused an allele dropout from at least one locus from three replicate measurements Data collection was performed on a 3130xl Genetic Analyzer and all collected data were analyzed using GeneMapper® ID Software version 3.2. RESULTS 150 uM Hematin 6 ug/ul Melanin = 0.0003N = 0.003N CONCLUSIONS The results of this study clearly demonstrated that different multiplex PCR assays can be differentially affected by the presence of various PCR inhibitors. AmpFℓSTR® MiniFilerTM PCR Amplification Kit, with enhanced PCR buffer formulation, optimized PCR primer sequences and improved thermal cycling parameters, noticeably outperformed AmpFℓSTR® Identifiler® under all inhibited PCR conditions. Inhibition of multiplex PCR assays can result in reduced product yield, complete failure, or unusual peak morphology (due to incomplete +A addition). During our investigation, we noticed the tendency for certain loci in AmpFℓSTR® MiniFilerTM to exhibit split-peak morphology under low pH PCR conditions. Subsequent analysis revealed that the split-peak morphology can be corrected by increasing the length of the final PCR extension step, without affecting other STR loci. REFERENCES AmpFℓSTR® Figure 1. High pH can affect PCR amplification In the presence of 0.004N acetic acid, incomplete +A addition was observed in AmpFℓSTR® MiniFilerTM PCR reactions. Significant reduction in incomplete +A addition was observed by increasing the final PCR extension time from 45 minutes at 60°C to 60 minutes. Further increase in final PCR extension time to 90 minutes effectively completed the +A addition. Identifiler® amplification Figure 4. Missing profiles in an inhibited can be recovered by AmpFℓSTR® MiniFilerTM at the same inhibitor concentration AmpFℓSTR® Identifiler® Missing D21, D7, CSF, D16, D2, D18, FGA 1. 2. 3. 4. 5. 6. 7. Akane, A et al. (1994) J. Forensic Sci. 39, 362-72. Shutler, G.G. et al. (1999) J. Forensic Sci. 44, 623-6. Eckhart, L. et al. (2000) Biochem. Biophys. Res. Comm. 271, 726-30. Tsai, Y.L. and Olson, B.H. (1992) Appl. Environ. Microbiol. 58, 2292-5. Kim, C.H. et al. (2001) J. Dairy Sci. 84, 74-83. Powell, H.A. et al. (1994) Lett. Appl. Microbiol. 18, 59-61. Chung, D.T. (2004) Ph.D. Thesis, Ohio University. ACKNOWLEDGEMENTS AmpFℓSTR® MiniFilerTM The performances of AmpFℓSTR® Identifiler® and AmpFℓSTR® MiniFilerTM in the presence of increasing concentration of NaOH were investigated. With AmpFℓSTR® Identifiler®, PCR inhibition was observed when NaOH is greater than 0.003N. On the contrary, full STR profiles were generated by AmpFℓSTR® MiniFilerTM even in the presence of 0.0055N NaOH. Hematin and melanin were prepared using NaOH solution and the highest concentrations of the two inhibitors used in this study are indicated on the graph. CSF D21 D18 D16 D2 D7 FGA In the presence of 1.25 mM of calcium, several loci in AmpFℓSTR® Identifiler® failed to amplify whereas AmpFℓSTR® MiniFilerTM generated a full STR profile. The STR profile generated by AmpFℓSTR® MiniFilerTM was able to recover all the missing alleles that AmpFℓSTR® Identifiler® failed to amplify. The authors wish to acknowledge the Human Identification Group for helpful discussions during the investigation. © 2008 Applied Biosystems