Application Note Human Identification AmpFlSTR Identifiler PCR Amplification Kit In Applied Biosystems continual efforts to improve the quality of our products, we have made some modifications to the manufacturing process of the AmpFlSTR Identifiler PCR Amplification Kit (PN 4322288) effective from lot number 0310018 onwards. For samples amplified with the Identifiler Kit, these modifications reduce the artifacts that are observed in both PET dye and VIC dye results (see Figure 1). A VIC dye labeled artifact has been observed at approximately 120 bp. In order to address this artifact, an additional step has been introduced in the purification process of the VIC dye labeled PCR primers in the Identifiler Kit. PET dye labeled artifacts have also been observed between the Amelogenin and the D5S818 loci. A modification to the manufacturing process of the PET dye labeled primers has been introduced, while maintaining the primer sequences, resulting in a reduction in the artifacts visualized. As noted in the Identifiler Kit User s Manual (PN 4323291), non-nucleotide linkers are used in primer synthesis between the primer oligonucleotides and the dye. The nonnucleotide linkers enable reproducible positioning of the alleles to facilitate inter-locus spacing. The PET dye artifacts were observed to be correlated with the use of a particular nonnucleotide linker. By using a different non-nucleotide linker during synthesis of the PET dye labeled primers in the Identifiler Kit primer set for this kit, the PET dye artifacts are now observed to be significantly diminished. The modifications made in the production of the PET and VIC dye labeled primers are reflected in both the Identifiler primer sets and the Identifiler Allelic Ladder. For this reason, it is important that the number 0310018 onwards, is used in conjunction with the supplied Allelic Ladder, and not an Allelic Ladder supplied with previously manufactured lots of the Identifiler Kit (0103001 to 0308017). VIC dye labeled artifact at ~120 bp PET dye labeled artifacts observed between the Amelogenin and the D5S818 loci Previously released Identifiler Kit, lot number 0301011 (representitive of lots 0103001 to 0308017). number 0310018. Figure 1. Comparison of the observed VIC dye labeled and PET dye labeled artifacts for negative control amplifications with a previously released number 0301011, and lot number 0310018. DNA was generated using the ABI PRISM 310 Genetic Analyzer with the Windows NT OS using the G5 module. The artifacts have been highlighted for illustrative purposes. Both the VIC dye labeled and PET dye labeled artifacts are greatly reduced in the bottom panels.
Validation Experiments Experiments to evaluate the performance of the Identifiler Kit produced with the updated manufacturing steps were performed at Applied Biosystems. Sensitivity, stability, reproducibility, precision, and accuracy were specifically addressed. Validation studies were undertaken to address the DNA Advisory Board s Quality Assurance Standards for Forensic DNA Testing and Scientific Working Group on DNA Analysis Methods Revised Validation Guidelines: (July 10, 2003) (unpublished). The developmental validation for the Identifiler Kit is thoroughly described in the Identifiler Kit User s Manual (PN 4323291), Previously released Identifiler Kit number 0310018 Chapter 4. Each laboratory using the Identifiler Kit should perform appropriate validation studies. Sensitivity Sensitivity of this kit was assessed by using both a previous lot (0103001 to 0308017) and a recent lot produced with the updated manufacturing process (lot number 0310018) of the Identifiler Kit, for the amplification of DNA samples with a range input concentrations from 0 ng to 1 ng. (see Figure 2). Assessing the results obtained for each dye used (6 FAM dye, blue; VIC dye, green; NED dye, yellow; PET dye, red) there was no significant difference in peak heights, Figure 2. Effect of amplifying various amounts of DNA ranging from 16 pg to 1 ng using a previous lot (0103001 to 0308017) and lot number 0310018 of the Identifiler Kit. Note that the y-scale is magnified for the lower amounts of DNA. DNA profiles were generated using the 310 Genetic Analyzer with the Macintosh OS, using the G5V2 module. Comparable sensitivity was obtained using the previous lot (0103001 to 0308017) and lot number 0310018 of the Identifiler Kit. as measured by relative fluorescence units (RFU) between kits. Data were generated using the ABI PRISM 310 Genetic Analyzer with the Macintosh Operating System and the ABI PRISM 3100 Genetic Analyzer. Results indicate that this kit provides comparable sensitivity to previously manufactured kits. Stability As with all fluorescent dye labeled amplicons used in conjunction with capillary electrophoresis platforms, dye artifacts have been observed to occur with the Identifiler Kit (see Identifiler Kit User s Manual, pages 4-25). Historically, these artifacts have been observed to be induced or worsened by inappropriate storage or handling. Therefore, it is important to follow the recommended protocols, storing the reagents at the recommended storage conditions, protecting reagents from light, minimizing handling, and not increasing activation or extension times. Stability studies were performed to determine the effects on the Identifiler Kit reagents, when subjected to environmental insults such as vigorous vortexing for 5 minutes prior to use and 24-hour exposure to laboratory ambient UV light at room temperature. Vortexing for 5 minutes prior to use did not have an observed effect on kit performance, however, 24-hour exposure to UV light at room temperature resulted in an increase in the VIC dye labeled artifact observed at ~120 bp, and an overall decrease in peak height. The effect of these conditions on the observation of artifacts and performance of the kit as measured by peak height, are shown in Figure 3. Similar results were observed using both the 310 Genetic Analyzer with the Macintosh OS and the 3100 Genetic Analyzer.
Reproducibility Reproducibility of this kit was assessed by amplification of DNA samples using both the previous lots (0103001 to 0308017) and a recent lot produced with the updated manufacturing process (lot number 0310018), of the Identifiler Kit. Samples were amplified using both the GeneAmp PCR System 9700 thermal cycler and the GeneAmp PCR System 9600 thermal cycler and analyzed using the 310 Genetic Analyzer with the Macintosh OS, the 310 Genetic Analyzer with the Windows NT OS, and the 3100 Genetic Analyzer (see Figure 4). Profiles generated were subsequently genotyped using the combination of GeneScan and Genotyper Software, and also with GeneMapper ID Software. Where profiles were generated with instrumentation using the 310 Genetic Analyzer with Macintosh OS, data was analyzed with GeneMapper ID Software using the Classic mode. Precision and Accuracy Sizing precision allows for accurate and reliable genotyping. Sizing precision was measured on the 3100 Genetic Analyzer and the 310 Genetic Analyzer with the Macintosh OS. In general, genotyping of the alleles uses +/-0.5 bp windows or bins around the size obtained for the alleles in the Identifiler Allelic Ladder. Precision results obtained from 10 repetitive injections of the allelic ladder, with both the 3100 Genetic Analyzer and the 310 Genetic Analyzer with the Macintosh OS are displayed in Table 1. These results were obtained within a single set of injections on a single capillary. For each allele, from the ten repetitive injections on the 3100 Genetic Analyzer and the 310 Genetic Analyzer with the Macintosh OS, a standard deviation in sizing of <0.25- bps was observed. GeneScan -500 LIZ size standard was used as the internal lane size standard. Figure 5 illustrates an electropherogram of the allelic ladder supplied with Identifiler Kit lot number 0310018, as generated using the 3100 Genetic Analyzer. It is important to note that while precision within a set of capillary injections is very good, the determined allele sizes may vary between platforms. For example, minor differences in sizing can be observed depending on type and concentration of polymer mixture, run temperature, and electrophoresis conditions. In summary, the sizing precision previously reported with the 310 Genetic Analyzer with the Macintosh OS (see Identifiler Kit User s Manual, Table 4-1) is statistically comparable to the results reported here. This document is the first reported results of sizing precision on the 3100 Genetic Analyzer. Overall, there is no significant difference observed in sizing precision as a result of manufacturing changes with the Identifiler Kit. Regular recommended storage. 24-hour storage at room temperature with light exposure. The two panels below are the expanded views of the dashed boxes highlighted above where the PET dye labeled artifacts between the Amelogenin and D5S818 loci, and the VIC dye labeled artifact at 120 bp reside. Regular recommended storage. 24-hour storage at room temperature with light exposure. Figure 3. Stability studies were conducted with lot number 0310018 of the Identifiler Kit. Our studies demonstrate that exposure to ambient laboratory UV light at room temperature for 24 hours reduced the overall peak heights and increased the VIC dye labeled artifact observed at ~120 bp. DNA profiles were generated using the 310 Genetic Analyzer with the Macintosh OS, using the G5V2 module.
Conclusion Minor modifications to the manufacturing process of the Identifiler Kit either significantly reduce or eliminate observed reproducible VIC dye labeled artifacts at approximately 120 bp and PET dye labeled artifacts between the Amelogenin and D5S818 loci. The reduction in observed reproducible VIC dye labeled and PET dye labeled related artifacts greatly facilitates the use of the Identifiler Kit for forensic casework involving mixed specimen samples. The two panels below are the expanded views of the dashed boxes highlighted above. Note the reduction of the PET dye labeled artifacts between the Amelogenin and D5S818 loci, and the VIC dye labeled artifact at 120 bp. Previously released number 0301001. number 0310018. Previously released number 0301011. number 0310018. Figure 4. Comparison of the profiles generated using previously released lot number 0301001 and lot number 0310018 of the Identifiler Kit with 1 ng of DNA. Comparable profiles were obtained with the different lots. Data was generated using the 310 Genetic Analyzer with the Windows NT OS using the G5 module. Figure 5. Electropherograms of the Identifiler Kit Allelic Ladder, lot number 0310018. Data was generated using the 3100 Genetic Analyzer.
Table 1. Examples of the precision results of ten injections of the AmpFlSTR Identifiler Allelic Ladder. ABI PRISM ABI PRISM 3100 Genetic 310 Genetic Analyzer Analyzer Allele Mean S.D. Mean S.D. Amelogenin X 106.49 0.07 107.34 0.07 Y 112.12 0.06 112.91 0.06 CSF1PO 6 304.60 0.10 305.44 0.12 7 308.89 0.11 309.91 0.17 8 313.13 0.13 314.30 0.17 9 317.35 0.14 318.61 0.17 10 321.49 0.15 322.83 0.15 11 325.63 0.15 326.99 0.17 12 329.72 0.14 331.07 0.14 13 333.75 0.14 335.13 0.16 14 337.74 0.13 339.09 0.13 15 341.61 0.12 342.58 0.09 D2S1338 15 307.09 0.13 308.16 0.16 16 311.38 0.12 312.63 0.13 17 315.63 0.13 316.97 0.17 18 319.81 0.12 321.27 0.17 19 323.96 0.13 325.47 0.17 20 328.08 0.15 329.61 0.16 21 332.14 0.14 333.68 0.14 22 336.23 0.14 337.71 0.16 23 340.21 0.13 341.45 0.12 24 343.85 0.11 344.84 0.06 25 347.51 0.07 348.18 0.05 26 351.16 0.05 351.65 0.04 27 354.90 0.06 355.29 0.04 28 358.71 0.05 358.95 0.05 D3S1358 12 111.55 0.07 112.37 0.08 13 115.63 0.04 116.43 0.06 14 119.57 0.05 120.35 0.08 15 123.52 0.07 124.29 0.06 16 127.71 0.06 128.45 0.07 17 131.91 0.07 132.60 0.06 18 135.97 0.07 136.66 0.07 19 140.09 0.06 140.80 0.06 D5S818 7 133.98 0.08 134.54 0.07 8 138.08 0.06 138.63 0.08 9 142.45 0.04 142.98 0.06 10 146.91 0.04 147.45 0.06 11 151.27 0.04 151.85 0.03 12 155.49 0.06 156.23 0.05 13 159.61 0.07 160.47 0.07 14 163.79 0.08 164.66 0.06 15 167.73 0.08 168.61 0.06 16 171.85 0.07 172.76 0.05 D7S820 6 255.00 0.06 255.59 0.08 7 259.04 0.06 259.60 0.08 8 263.06 0.06 263.68 0.08 9 267.11 0.08 267.74 0.10 10 271.15 0.07 271.78 0.11 11 275.19 0.07 275.89 0.10 12 279.27 0.07 279.97 0.10 13 283.29 0.09 284.00 0.10 14 287.40 0.07 288.09 0.12 15 291.45 0.07 292.20 0.09 D8S1179 8 122.98 0.06 123.68 0.07 9 127.05 0.07 127.70 0.05 10 131.12 0.06 131.71 0.07 11 135.23 0.05 135.82 0.08 12 139.45 0.06 140.04 0.07 13 144.03 0.05 144.62 0.07 14 148.49 0.05 149.13 0.05 15 152.87 0.05 153.60 0.04 16 157.12 0.06 158.04 0.06 17 161.30 0.08 162.27 0.07 18 165.39 0.09 166.39 0.07 19 169.46 0.08 170.51 0.07 ABI PRISM ABI PRISM 3100 Genetic 310 Genetic Analyzer Analyzer Allele Mean S.D. Mean S.D. D13S317 8 216.60 0.07 217.24 0.06 9 220.57 0.08 221.20 0.08 10 224.53 0.08 225.17 0.05 11 228.61 0.06 229.30 0.07 12 232.53 0.08 233.20 0.07 13 236.43 0.07 237.12 0.08 14 240.44 0.07 241.11 0.10 15 244.43 0.08 245.13 0.08 D16S539 5 252.26 0.08 252.72 0.08 8 264.20 0.05 264.73 0.10 9 268.23 0.07 268.75 0.10 10 272.22 0.09 272.80 0.09 11 276.29 0.08 276.86 0.10 12 280.27 0.09 280.87 0.11 13 284.28 0.09 284.90 0.11 14 288.37 0.10 289.00 0.09 15 292.42 0.10 293.10 0.11 D18S51 7 261.79 0.07 262.41 0.05 9 269.95 0.06 270.61 0.08 10 274.06 0.06 274.75 0.10 10.2 276.06 0.06 276.78 0.08 11 278.14 0.06 278.90 0.07 12 282.25 0.08 283.03 0.07 13 286.36 0.07 287.16 0.11 13.2 288.38 0.05 289.20 0.11 14 290.48 0.08 291.32 0.11 14.2 292.49 0.07 293.39 0.12 15 294.60 0.09 295.50 0.11 16 298.74 0.08 299.71 0.12 17 303.10 0.11 304.36 0.13 18 307.52 0.11 308.98 0.14 19 311.85 0.13 313.49 0.18 20 316.17 0.13 317.94 0.18 21 320.42 0.14 322.24 0.19 22 324.63 0.14 326.50 0.17 23 328.80 0.14 330.68 0.17 24 332.93 0.13 334.82 0.17 25 337.02 0.14 338.86 0.17 26 341.00 0.13 342.48 0.10 27 344.70 0.10 345.91 0.06 D19S433 9 101.71 0.04 102.26 0.05 10 105.59 0.02 106.12 0.04 11 109.50 0.05 109.97 0.04 12 113.40 0.05 113.86 0.05 12.2 115.40 0.04 115.81 0.04 13 117.36 0.03 117.75 0.04 13.2 119.36 0.05 119.71 0.04 14 121.31 0.03 121.66 0.06 14.2 123.34 0.03 123.63 0.04 15 125.28 0.04 125.57 0.05 15.2 127.31 0.04 127.59 0.06 16 129.30 0.05 129.56 0.04 16.2 131.35 0.05 131.53 0.04 17 133.35 0.05 133.50 0.05 17.2 135.39 0.03 135.53 0.06 D21S11 24 184.56 0.06 185.20 0.04 24.2 186.54 0.05 187.17 0.05 25 188.50 0.05 189.11 0.05 26 192.45 0.06 192.99 0.04 27 196.37 0.04 196.87 0.05 28 200.25 0.04 200.69 0.03 28.2 202.19 0.05 202.61 0.04 29 204.16 0.04 204.60 0.04 29.2 206.12 0.04 206.54 0.05 30 208.14 0.03 208.53 0.05 30.2 210.08 0.05 210.49 0.03 31 212.08 0.03 212.43 0.04 31.2 214.03 0.04 214.39 0.04 32 216.04 0.05 216.38 0.06 ABI PRISM ABI PRISM 3100 Genetic 310 Genetic Analyzer Analyzer Allele Mean S.D. Mean S.D. 32.2 218.01 0.05 218.33 0.04 33 220.04 0.04 220.33 0.04 33.2 221.96 0.04 222.28 0.04 34 224.02 0.04 224.34 0.07 34.2 225.93 0.05 226.26 0.06 35 227.99 0.05 228.31 0.05 35.2 229.92 0.05 230.23 0.06 36 231.95 0.05 232.25 0.06 37 235.94 0.04 236.23 0.06 38 239.85 0.04 240.15 0.05 FGA 17 214.46 0.07 215.31 0.06 18 218.44 0.09 219.30 0.07 19 222.46 0.08 223.33 0.08 20 226.46 0.08 227.32 0.08 21 230.48 0.08 231.34 0.07 22 234.50 0.08 235.37 0.10 23 238.53 0.09 239.42 0.09 24 242.57 0.08 243.45 0.07 25 246.61 0.08 247.51 0.11 26 250.66 0.07 251.59 0.11 26.2 252.68 0.08 253.64 0.09 27 254.69 0.09 255.63 0.11 28 258.74 0.06 259.69 0.11 29 262.83 0.10 263.82 0.12 30 266.92 0.09 267.96 0.12 30.2 268.75 0.09 269.80 0.12 31.2 272.82 0.09 273.92 0.12 32.2 276.93 0.11 278.05 0.13 33.2 281.03 0.11 282.19 0.13 42.2 319.28 0.17 321.45 0.20 43.2 323.49 0.17 325.74 0.22 44.2 327.67 0.17 329.91 0.19 45.2 331.84 0.18 334.08 0.20 46.2 335.83 0.17 338.02 0.19 47.2 339.90 0.17 341.74 0.14 48.2 343.65 0.13 345.21 0.09 50.2 351.06 0.13 352.18 0.06 51.2 354.83 0.10 355.80 0.07 TH01 4 162.96 0.07 163.67 0.04 5 167.02 0.04 167.74 0.06 6 171.06 0.07 171.78 0.04 7 175.06 0.07 175.79 0.05 8 179.07 0.06 179.79 0.04 9 183.05 0.06 183.76 0.05 9.3 186.06 0.06 186.75 0.05 10 186.97 0.06 187.67 0.04 11 190.93 0.04 191.55 0.06 13.3 201.74 0.05 202.21 0.05 TPOX 6 221.96 0.05 222.32 0.07 7 225.92 0.05 226.25 0.08 8 229.84 0.05 230.20 0.06 9 233.82 0.05 234.13 0.05 10 237.76 0.07 238.10 0.05 11 241.76 0.07 242.10 0.05 12 245.73 0.06 246.06 0.06 13 249.73 0.04 250.08 0.06 vwa 11 154.24 0.05 154.99 0.06 12 158.43 0.06 159.32 0.05 13 162.57 0.07 163.49 0.05 14 166.79 0.06 167.72 0.05 15 170.70 0.08 171.65 0.06 16 174.71 0.07 175.63 0.07 17 178.70 0.09 179.63 0.05 18 182.67 0.10 183.56 0.05 19 186.60 0.09 187.46 0.05 20 190.54 0.06 191.33 0.04 21 194.47 0.08 195.22 0.05 22 198.34 0.07 199.02 0.05 23 202.18 0.07 202.80 0.06 24 206.44 0.06 207.06 0.07
iscience. To achieve accurate, reproducible results, life scientists are taking advantage of advanced analysis systems that unite technology, informatics, and traditional laboratory research. In partnership with our customers, Applied Biosystems provides the innovative products, services, and knowledge resources that make this new, Integrated Science possible. Worldwide Sales Offices Applied Biosystems vast distribution and service network, composed of highly trained support and applications personnel, reaches 150 countries on six continents. For international office locations, please call the division headquarters or refer to our Web site at Applera is committed to providing the world s leading technology and information for life scientists. Applera Corporation consists of the Applied Biosystems and Celera Genomics businesses. Headquarters 850 Lincoln Centre Drive Foster City, CA 94404 USA Phone: 650.638.5800 Toll Free: 800.345.5224 Fax: 650.638.5884 For Research, Forensic, or Paternity Use Only. Not for use in diagnostic procedures. AB (Design), FAM, iscience, iscience (Design), and NED are trademarks, and ABI PRISM and its design, AmpFlSTR, Applied Biosystems, Applera, GeneAmp, Genotyper, Identifiler, GeneScan, GeneMapper, PET, VIC, and LIZ are registered trademarks of Applera Corporation or its subsidiaries in the US and/or certain other countries. 2004. All Rights Reserved. Printed in the USA, 06/04 P+s Publication 112AP02-02