TECHNICAL MANUAL GenePrint® STR Systems (Silver Stain Detection) InstrucƟons for use of Products DC1191, DC4001, DC4011, DC4021, DC4031, DC4041, DC4051, DC4061, DC4061, DC4071, DC4080, DC4081, DC6000, DC6001, DC6031, DC6451, DG2141 and DG2101 Revised 10/15 TMD004 GenePrint ® STR Systems (Silver Stain Detection) All technical literature is available on the Internet at: www.promega.com/protocols Please visit the web site to verify that you are using the most current version of this Technical Manual. Please contact Promega Technical Services if you have questions on use of this system. E-mail: [email protected] 1. Description..................................................................................................................................2 2. Product Components and Storage Conditions ....................................................................2 3. Before You Begin .......................................................................................................................4 4. Amplification .............................................................................................................................5 A. Choice of Thermal Cycling Protocol .............................................................................5 B. Amplification Setup.........................................................................................................9 C. Amplification Thermal Cycling ...................................................................................11 5. Polyacrylamide Gel Preparation...........................................................................................12 A. Notes ................................................................................................................................12 B. Procedure.........................................................................................................................13 6. Polyacrylamide Gel Electrophoresis....................................................................................15 A. Gel Pre-Run.....................................................................................................................15 B. Sample Preparation........................................................................................................15 C. Sample Loading..............................................................................................................16 D. Gel Electrophoresis ........................................................................................................16 7. Silver Staining..........................................................................................................................17 A. Procedure.........................................................................................................................17 B. Reuse of Glass Plates .....................................................................................................18 8. Exposure of Film......................................................................................................................19 9. Data Analysis ...........................................................................................................................20 A. pGEM® DNA Markers...................................................................................................21 B. Controls ...........................................................................................................................21 C. STR Ladders ....................................................................................................................21 10. Representative STR Data .......................................................................................................22 11. Troubleshooting.......................................................................................................................26 12. References .................................................................................................................................28 13. Appendix ...................................................................................................................................30 A. Advantages of STR Typing...........................................................................................30 B. Advantages of Using the Loci in the GenePrint ® STR Systems...............................30 C. Power of Discrimination ...............................................................................................34 D. DNA Extraction and Quantitation Methods..............................................................35 E. Agarose Gel Electrophoresis of Amplification Products (Optional)......................36 F. Composition of Buffers and Solutions........................................................................37 G. Population Data..............................................................................................................38 H. Organizational Sheets....................................................................................................39 I. Related Products ............................................................................................................42 14. Summary of Changes..............................................................................................................43 Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com Printed in USA. Revised 10/15 Part# TMD004 Page 1 1. Description STR (short tandem repeat) loci consist of short, repetitive sequence elements 3–7 base pairs in length (1–4). These repeats are well distributed throughout the human genome and are a rich source of highly polymorphic markers, which often may be detected using PCR (5–8). Alleles of these loci are differentiated by the number of copies of the repeat sequence contained within the amplified region and are distinguished from one another using silver stain or fluorescent detection following electrophoretic separation. The GenePrint ® STR Systems provide all of the materials required to perform 100 or 400 amplification reactions except for Taq DNA polymerase and sample DNA. Accessory components are available to simplify many of the procedures related to STR analysis (Section 13.I). This manual describes methods we have evaluated and recommend for sample preparation, amplification of sample, separation of amplified products and silver detection of separated material. All of the GenePrint ® STR Systems can be amplified using either the Perkin-Elmer model 480 or 9600 thermal cyclers, but slight differences in yield or balance between loci might be observed if the system is not first optimized on that particular thermal cycler. Information about allele frequencies for African-Americans, Caucasian-Americans and Hispanic-Americans for all currently available STR Systems is available in Section 13.G. Additional population data for STR loci can be found in Edwards et al. (3), Puers et al. (9), Hammond et al. (10), Bever et al. (11), Sprecher et al. (12) and Lins et al. (13). 2. Product Components and Storage Conditions GenePrint ® STR Multiplex Systems (Silver Stain Detection) Each system contains the appropriate locus-specific primer pair and allelic ladder in addition to STR 10X Buffer, K562 DNA, STR 2X Loading Solution and pGEM® DNA Markers. Multiplex STR Systems include a single tube containing all required 10X primer pairs as a mixture for simultaneous amplification of more than one locus and another tube containing a mixture of the allelic ladders for the same set of loci. Product GenePrint ® SilverSTR® III System(a) Size Cat.# 100 reactions DC6451 400 reactions DC6450 Not For Medical Diagnostic Use. Cat.# DC6450 contains sufficient reagents for 400 reactions of 25µl each. Includes: • • • • • • • 1ml 600µl 1.2ml 3µg 2ml 3µg 1 SilverSTR® III 10X Primer Pair Mix SilverSTR® III Allelic Ladder Mix STR 10X Buffer K562 DNA High Molecular Weight (10ng/µl) STR 2X Loading Solution pGEM® DNA Markers (20ng/µl) Protocol Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com Part# TMD004 Page 2 Printed in USA. Revised 10/15 2. Product Components and Storage Conditions (continued) Product GenePrint ® STR Multiplex System— CSF1PO, TPOX, TH01 Multiplex(a) GenePrint ® STR Multiplex System— F13A01, FESFPS, vWA Multiplex Size Cat.# 100 reactions 400 reactions DC6001 DC6000 100 reactions 400 reactions DC6031 DC6030 Not For Medical Diagnostic Use. GenePrint ® STR Monoplex Systems (Silver Stain Detection) Each system contains the specific primer pair and ladder plus other components sufficient to perform the specified number of reactions. Product GenePrint ® Sex Identification System Amelogenin (Silver Detection) GenePrint ® STR System—CSF1PO GenePrint ® STR System—F13A01 GenePrint ® STR System—F13B GenePrint ® STR System—FESFPS GenePrint ® STR System—HPRTB GenePrint ® STR System—LPL GenePrint ® STR System—TH01 GenePrint ® STR System—TPOX GenePrint ® STR System—vWA Not For Medical Diagnostic Use. Size Cat.# 100 reactions 100 reactions 100 reactions 100 reactions 100 reactions 100 reactions 100 reactions 100 reactions 100 reactions 100 reactions DC4081 DC4011 DC4041 DC4001 DC4021 DC4061 DC4071 DC1191 DC4051 DC4031 Size 150µl 150µl Cat.# DG2101 DG2141 Allelic Ladders(a) Product CTT Allelic Ladder Mix FFv Allelic Ladder Mix Storage Conditions: Store all components at –20°C. The pre- and postamplification components (Allelic Ladder Mix, STR 2X Loading Solution and pGEM® DNA Markers) are sealed in separate packages to prevent crosscontamination. We strongly recommend that pre-amplification and postamplification reagents be stored separately and handled with different pipettes, tube racks, etc. Store amplified material at –20°C. Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com Printed in USA. Revised 10/15 Part# TMD004 Page 3 3. Before You Begin The application of PCR-based typing for forensic or paternity casework requires validation studies and quality-control measures that are not contained in this manual (14,15). The quality of the purified DNA sample, as well as small changes in buffers, ionic strength, primer concentrations, choice of thermal cycler and thermal cycling conditions, can affect the success of amplification. We suggest strict adherence to recommended procedures for amplification, denaturing gel electrophoresis, silver stain analysis and recording of data on film. PCR-based STR analysis is subject to contamination by very small amounts of nontemplate human DNA. Extreme care should be taken to avoid cross-contamination when preparing sample DNA, handling primer pairs, setting up amplification reactions and analyzing amplification products. Reagents and materials used prior to amplification (STR 10X Buffer, K562 Control DNA and 10X Primer Pairs) are provided in a separate box and should be stored separately from those used following amplification (Allelic Ladders, STR 2X Loading Solution and pGEM® DNA Markers). Always include a negative control reaction (i.e., no template) to detect reagent contamination. We highly recommend the use of gloves and aerosol-resistant pipet tips. Some of the reagents used in the analysis of STR products are potentially hazardous and should be handled accordingly. Table 1 describes the potential hazards associated with such reagents. Table 1. Hazardous Reagents. Reagent Hazard acetic acid (fix/stop solution) corrosive, hygroscopic acrylamide ammonium persulfate suspected carcinogen, toxic oxidizer, corrosive bisacrylamide toxic, irritant formaldehyde (staining solution and developer solution) formamide (STR 2X Loading Solution) highly toxic, suspected carcinogen irritant, teratogen methacryloxypropyltrimethoxysilane (bind silane) toxic, moisture sensitive silver nitrate (staining solution) highly toxic, oxidizer sodium thiosulfate (developer solution) irritant, hygroscopic TEMED corrosive, flammable urea irritant xylene cyanol FF (STR 2X Loading Solution) irritant Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com Part# TMD004 Page 4 Printed in USA. Revised 10/15 4. Amplification The GenePrint ® STR Systems have been developed for amplification without artifacts using standard Taq DNA polymerase. Special enzymes such as AmpliTaq Gold® DNA polymerase are not required for peak performance. However, if using AmpliTaq Gold® DNA polymerase, use the GoldST*R 10X Buffer (Cat.# DM2411, available separately) instead of the STR 10X Buffer. The STR 10X Buffer (pH 9.0) is not compatible with AmpliTaq Gold® DNA polymerase because the modified Taq DNA polymerase is optimized at pH 8.3. Also, when using AmpliTaq Gold® DNA polymerase, incorporate an additional incubation at 95°C for 11 minutes prior to initiation of the thermal cycling program. Materials to Be Supplied by the User (Solution compositions are provided in Section 13.F.) • thermal cycler, model 480 or GeneAmp® system 9600 (Perkin-Elmer) • microcentrifuge • Taq DNA polymerase • Nuclease-Free Water (Cat.# P1193 or equivalent) • Mineral Oil (Cat.# DY1151 or equivalent) • 0.5ml or 0.2ml microcentrifuge tubes (compatible with thermal cycler) • 1.5ml microcentrifuge tubes • BSA Fraction V (optional) • aerosol-resistant pipet tips • crushed ice 4.A. Choice of Thermal Cycling Protocol The CTT and FFv multiplexes, their corresponding monoplexes, the GenePrint ® Sex Identification System—Amelogenin and the GenePrint ® STR System—HPRTB are optimized for use with Perkin-Elmer GeneAmp® reaction tubes and the Perkin-Elmer model 480 thermal cycler. The SilverSTR® III System is optimized for use with MicroAmp® tubes and the GeneAmp® PCR system 9600 thermal cycler. However, each system may be used with either thermal cycler. Please refer to Tables 2 and 3 for recommended and alternative protocols for each system and thermal cycler. The cycling conditions for each protocol are given in Table 4. When using a thermal cycler on which a system was not optimized, there may be a small loss in product yield or sensitivity, and the balance between loci may change slightly in the multiplex systems. Meticulous care must be taken to ensure successful amplification. A guide to amplification troubleshooting is provided in Section 11. Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com Printed in USA. Revised 10/15 Part# TMD004 Page 5 Table 2. Protocol Options for the Model 480 Thermal Cycler. Recommended Protocols1 Alternative Protocols2 CTT Multiplex 2 1 CTT Multiplex with Amelogenin 2 1 FFv Multiplex 7 1 7 NA Amelogenin 2 1 CSF1PO, F13A01, TH01 or TPOX 2 NA F13B, LPL or vWA 7 1 FESFPS or HPRTB 1 NA GenePrint ® STR System Multiplexes SilverSTR® III Multiplex3 Individual Systems NA = not applicable. 1Recommended protocols offer similar performance characteristics. 2Alternative protocols also work but may trade off performance characteristics, such as greater speed or convenience, for less sensitivity. 3Performance variation of thermal cyclers may cause extraneous bands to be generated above the allele range. See Section 9. Table 3. Protocol Options for the GeneAmp® PCR System 9600 Thermal Cycler. Recommended Protocols1 Alternative Protocols2 CTT Multiplex 5,6 12 CTT Multiplex with Amelogenin 5,6 12 FFv Multiplex 8,9 3,4 SilverSTR® III Multiplex3 10 NA Individual Systems Amelogenin, CSF1PO, F13A01, TH01 or TPOX 5,6 NA F13B, FESFPS, HPRTB, LPL or vWA 3,4 NA GenePrint ® STR System Multiplexes NA = not applicable. 1Recommended protocols offer similar performance characteristics. 2Alternative protocols also work but may trade off performance characteristics, such as greater speed or convenience, for less sensitivity. 3Performance variation of thermal cyclers may cause extraneous bands to be generated above the allele range. See Section 9. Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com Part# TMD004 Page 6 Printed in USA. Revised 10/15 Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com Printed in USA. Revised 10/15 Part# TMD004 Page 7 Protocol Number 1 (Refer to Note 1) 2 (Refer to Note 1) 3 (Refer to Note 2) 4 (Refer to Note 3) 5 (Refer to Note 2) 6 (Refer to Note 3) 7 (Refer to Note 1) 8 (Refer to Note 2) Initial Incubation2 96°C for 2 minutes 96°C for 2 minutes 96°C for 2 minutes 96°C for 2 minutes 96°C for 2 minutes 96°C for 2 minutes 96°C for 2 minutes 96°C for 2 minutes Thermal Cycler1 480 480 9600 9600 9600 9600 480 9600 Table 4. Amplification Protocols. Cycling for First 10 Cycles 94°C, 1 minute 60°C, 1 minute 70°C, 1.5 minutes None 94°C, 1 minute 64°C, 1 minute 70°C, 1.5 minutes None 94°C, 1 minute 60°C, 1 minute 70°C, 1.5 minutes 50 seconds to 94°C, 1 minute 34 seconds to 60°C, 1 minute 25 seconds to 70°C, 1.5 minutes None 94°C, 1 minute 64°C, 1 minute 70°C, 1.5 minutes 50 seconds to 94°C, 1 minute 30 seconds to 64°C, 1 minute 15 seconds to 70°C, 1.5 minutes None 94°C, 1 minute 60°C, 1 minute 70°C, 1.5 minutes None 94°C, 1minute 60°C, 1 minute 70°C, 1.5 minutes Programmed Ramp Times None Cycling for Last 20 Cycles 90°C, 1 minute 60°C, 1 minute 70°C, 1.5 minutes None 90°C, 1 minute 64°C, 1 minute 70°C, 1.5 minutes None 90°C, 1 minute 60°C, 1 minute 70°C, 1.5 minutes 45 seconds to 90°C, 1 minute 30 seconds to 60°C, 1 minute 25 seconds to 70°C, 1.5 minutes None 90°C, 1 minute 64°C, 1 minute 70°C, 1.5 minutes 45 seconds to 90°C, 1 minute 26 seconds to 64°C, 1 minute 15 seconds to 70°C, 1.5 minutes None 90°C, 1 minute 60°C, 1 minute 70°C, 1.5 minutes None 90°C, 1 minute 60°C, 1 minute 70°C, 1.5 minutes Programmed Ramp Times None 60°C for 30 minutes 60°C for 30 minutes None None None None None Extension Step None 4°C 4°C 4°C 4°C 4°C 4°C 4°C Hold Step 4°C Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com Part# TMD004 Page 8 Printed in USA. Revised 10/15 Initial Incubation2 96°C for 2 minutes 96°C for 1 minute 96°C for 2 minutes 96°C for 2 minutes Thermal Cycler1 9600 9600 9600 9600 Programmed Cycling for Ramp Times First 10 Cycles 50 seconds to 94°C, 30 seconds 34 seconds to 60°C, 1 minute 25 seconds to 70°C, 1.5 minutes Default ramp to 94°C, 30 seconds 68 seconds to 60°C, 30 seconds 50 seconds to 70°C, 45 seconds 50 seconds to 94°C, 1 minute 34 seconds to 60°C, 1 minute 25 seconds to 70°C, 1.5 minutes 50 seconds to 94°C, 1 minute 30 seconds to 64°C, 1 minute 15 seconds to 70°C, 1.5 minutes Programmed Cycling for Ramp Times Last 20 Cycles 45 seconds to 90°C, 1 minute 30 seconds to 60°C, 1 minute 25 seconds to 70°C, 1.5 minutes Default ramp to 90°C, 30 seconds 60 seconds to 60°C, 30 seconds 50 seconds to 70°C, 45 seconds 45 seconds to 90°C, 1 minute 30 seconds to 60°C, 1 minute 25 seconds to 70°C, 1.5 minutes 45 seconds to 90°C, 1 minute 26 seconds to 64°C, 1 minute 15 seconds to 70°C, 1.5 minutes None 60°C for 30 minutes 60°C for 30 minutes Extension Step 60°C for 30 minutes 4°C 4°C 4°C Hold Step 4°C refers to the Perkin-Elmer model 480 thermal cycler; 9600 refers to the Perkin-Elmer GeneAmp® PCR system 9600 thermal cycler. 2Initial incubation performed using AmpliTaq® DNA polymerase. When using AmpliTaq Gold® DNA polymerase, include an additional incubation at 95°C for 11 minutes prior to initiation of the thermal cycling program. Also when using AmpliTaq® DNA polymerase, be sure to use the GoldST*R 10X Buffer. 1480 Protocol Number 9 (Refer to Note 3) 10 (Refer to Note 4) 11 (Refer to Note 4) 12 (Refer to Note 4) Table 4. Amplification Protocols (continued). Notes for Table 4: 1. Use GeneAmp® reaction tubes, and overlay all reactions with mineral oil. 2. Use GeneAmp® reaction tubes in combination with the GeneAmp® thinwalled tray. This reduces the maximum number of simultaneous reactions to 48 due to the spacing of holes in the tray. Add mineral oil to all reactions. 3. Use MicroAmp® reaction tubes in the MicroAmp® 9600 tray. This allows a maximum of 96 simultaneous reactions. Add mineral oil to all reactions. not cover the reactions with the system 9600 thermal cycler lid. Cover ! Do the reaction tubes loosely with aluminum foil. Optional: Add BSA Fraction V (final concentration 60µg/ml) to all reactions. This may result in a slight increase in yield. It will also produce higher silver background in the gel lanes. We recommend Sigma BSA (Cat.# A2153). Performance may vary depending on the source of this component. 4. Use MicroAmp® reaction tubes in the MicroAmp® 9600 tray. This allows a maximum of 96 simultaneous reactions. No mineral oil is needed. ! Cover reactions with the system 9600 thermal cycler lid. Optional: Add BSA Fraction V (final concentration 60µg/ml) to all reactions. This may result in a slight increase in yield. It will also produce higher silver background in the gel lanes. We recommend Sigma BSA (Cat.# A2153). Performance may vary depending on the source of this component. 4.B. Amplification Setup The use of gloves and aerosol-resistant pipet tips is highly recommended to prevent cross-contamination. Helpful organizational sheets are provided in Section 13.H. 1. Thaw the STR 10X Buffer and 10X Primer Pair(s), and place on ice. Notes: 1. Mix reagents by vortexing for 15 seconds before each use. 2. If using AmpliTaq Gold® DNA polymerase, use the GoldST*R 10X Buffer (Cat.# DM2411) instead of the STR 10X Buffer. 2. Place one clean, autoclaved 0.5ml reaction tube for each reaction into a rack, and label appropriately. Note: If using the GeneAmp® PCR system 9600 thermal cycler, refer to the notes for Table 4 for tube selection. 3. Determine the number of reactions to be set up. This should include a positive and negative control reaction. Add 1 or 2 reactions to this number to compensate for pipetting error. While this approach does waste a small amount of each reagent, it ensures that you will have enough PCR master mix for all samples. Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com Printed in USA. Revised 10/15 Part# TMD004 Page 9 4.B. Amplification Setup (continued) 4. Calculate the required amount of each component of the PCR master mix (Table 5). Multiply the volume (µl) per sample by the total number of reactions (from Step 3) to obtain the final volume (µl). Note: The CTT Multiplex and Amelogenin locus can be amplified simultaneously. 5. In the order listed in Table 5, add the final volume of each reagent to a sterile tube. Mix gently (do not vortex), and place on ice. Note: The volume given assumes a Taq DNA polymerase concentration of 5u/µl. For different enzyme concentrations, the volume of enzyme added must be adjusted accordingly. If the final volume of Taq DNA polymerase added to the master mix is less than 0.5µl, you may wish to dilute the enzyme with STR 1X Buffer, and add a larger volume. The amount of sterile water should be adjusted accordingly so that the final volume per reaction is 25µl. Do not store diluted Taq DNA polymerase. Table 5. PCR Amplification Reaction Setup. Multiplex Reactions Containing Three Loci PCR Master Mix Component sterile water STR 10X Buffer Multiplex 10X Primer Pair Mix Taq DNA polymerase (at 5u/µl) total volume Volume Per Sample (µl) 17.35 2.50 2.50 0.15 (0.75u) 22.50 × Number of Final = Reactions Volume (µl) Combined CTTv Multiplex and Amelogenin Reactions PCR Master Mix Component sterile water STR 10X Buffer CTT Multiplex 10X Primer Pair Mix Amelogenin 10X Primer Pair Taq DNA polymerase (at 5u/µl) total volume Volume Per Sample (µl) 14.85 2.50 2.50 2.50 0.15 (0.75u) 22.50 × Number of Final = Reactions Volume (µl) Monoplex or Amelogenin-Only Reactions PCR Master Mix Component sterile water STR 10X Buffer locus-specific 10X primer pair Taq DNA polymerase (at 5u/µl) total volume Volume Per Sample (µl) 17.45 2.50 2.50 0.05 (0.25u) 22.50 × Number of Reactions = Final Volume (µl) Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com Part# TMD004 Page 10 Printed in USA. Revised 10/15 6. ! 7. Add 22.5µl of PCR master mix to each tube, and place on ice. Failure to keep the reagents and samples on ice can produce imbalanced amplification of multiplexed loci. If using AmpliTaq Gold® DNA polymerase, it is not necessary to keep the reactions on ice. Pipet 2.5µl of each sample into the respective tube containing 22.5µl of PCR master mix. Notes: 1. For the multiplex CTT and SilverSTR® III Systems, use 1–5ng of template DNA. For the multiplex FFv System, use 5–10ng of template DNA. 2. If the template DNA is stored in TE buffer (10mM Tris-HCl, 1mM EDTA [pH 7.5]), the volume of the DNA sample added should not exceed 20% of the final reaction volume. PCR amplification efficiency and quality can be altered greatly by changes in pH (due to added Tris-HCl) or available magnesium concentration (due to chelation by EDTA). DNA samples stored or diluted in sterile, deionized water are not subject to this caution. 8. For the positive amplification control, pipet 2.5µl (5ng) of K562 DNA (diluted to 2ng/µl) into a 0.5ml reaction tube containing 22.5µl of PCR master mix. 9. For a negative amplification control, pipet 2.5µl of sterile water (instead of template DNA) into a 0.5ml reaction tube containing 22.5µl of PCR master mix. 10. If recommended by the cycling protocol, add 1 drop of mineral oil to each tube. Close the tubes. Note: Allow the mineral oil to flow down the side of the tube and form an overlay to limit sample loss or cross-contamination due to splattering. 11. Centrifuge the samples briefly to bring the contents to the bottom of the tube. 4.C. Amplification Thermal Cycling 1. Place the tubes in a thermal cycler. 2. Select and run a recommended protocol from Table 2 or 3 (Section 4.A). 3. After completion of the thermal cycling protocol, store the samples at –20°C. Note: Storage of amplified samples at 4°C or above may produce degradation products. Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com Printed in USA. Revised 10/15 Part# TMD004 Page 11 5. Polyacrylamide Gel Preparation Materials to Be Supplied by the User (Solution compositions are provided in Section 13.F.) • 40% acrylamide:bis (19:1) and TEMED • 10X TBE Buffer (Cat.# V4251) • 10% Ammonium Persulfate (Cat.# V3131) • Urea (Cat.# V3171) • bind silane (methacryloxypropyltrimethoxysilane) • Gel Slick® solution (Cambrex Cat.# 50640) • 0.5% acetic acid in 95% ethanol • Nalgene® tissue culture filter (0.2 micron) • polyacrylamide gel electrophoresis apparatus for gels ≥30cm (e.g., SA32 or S2) • glass plates and side spacers for polyacrylamide gel ≥30cm • 14cm vinyl doublefine sharkstooth comb(s), 49 point, 0.4mm thick; or square-tooth comb, 35cm, 60 wells (cut in half for 30 wells/gel), 0.4mm thick (Owl Scientific Cat.# S2S-60A) • power supply • Liqui-Nox® detergent (Use of Liqui-Nox® detergent is extremely important, as other kinds of detergent can build up on the glass plates.) • clamps (e.g., large office binder clips) • diamond pencil for marking glass plates 5.A. Notes 1. Use 6% acrylamide for the GenePrint ® SilverSTR® III System. In a 4% gel, DNA strand separation in the locus D16S539 are such that the top strand of one allele overlaps with the bottom strand of the next larger allele. See Figure 2. 2. Unpolymerized acrylamide is a neurotoxin and suspected carcinogen; avoid inhalation and contact with skin. Read the warning label, and take the necessary precautions when handling this substance. Always wear gloves and safety glasses when working with acrylamide powder or solutions. 3. Bind silane is toxic and should be used in a chemical fume hood. 4. The longer glass plate will be treated with Gel Slick® solution to prevent the gel from sticking, and the shorter glass plate will be treated with bind silane to bind the gel. The two plates must be kept apart at all times to prevent cross-contamination. 5. All cleaning utensils (sponges) for the longer glass plates should be kept separate from those for the shorter glass plates to prevent crosscontamination of the binding solution. 6. The shorter glass plate preparation must be repeated for each gel. The longer glass plate preparation must be repeated after every four gels. Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com Part# TMD004 Page 12 Printed in USA. Revised 10/15 7. To remove the glass plate treatments (Gel Slick® solution or bind silane) immerse the plate(s) in 10% NaOH solution for 1 hour. Thoroughly rinse the plate(s) with deionized water, and clean with a detergent. The same 10% NaOH solution may be used for multiple gels. 8. New glass plates should be soaked in 10% NaOH for 1 hour and then rinsed thoroughly with deionized water before use. New plates also should be etched with a diamond pencil in the corner of one side to distinguish the sides of the plates in contact with the gel. 5.B. Procedure The following protocol is for the preparation of a denaturing polyacrylamide gel with the dimensions of 31.0cm wide × 38.5cm high × 0.4mm thick (e.g., S2 sequencing gel electrophoresis apparatus, Whatman Cat.# 21105-010). Use onehalf of the volumes described here for a gel with the dimensions of 17cm wide × 32cm high × 0.4mm thick (e.g., SA32 sequencing gel apparatus, Whatman Cat.# 31096-019). 1. Etch each glass plate on one side in one corner with a diamond pencil to distinguish the treated sides of the glass plates. Thoroughly clean the shorter and longer glass plates twice with 95% ethanol and Kimwipes® tissues. Note: The gel side is the etched side of the glass plate. 2. Using gloves, apply 3ml of Gel Slick® solution onto the etched side of the longer glass plate. With a dry paper towel, spread the Gel Slick® solution using a circular motion over the entire surface. 3. Wait 5 minutes for the Gel Slick® solution to dry. Remove the excess Gel Slick® solution with a paper towel saturated with deionized water. Finally, dry the glass plate with Kimwipes® tissue. 4. In a chemical fume hood, prepare fresh binding solution by adding 3µl of bind silane to 1ml of 0.5% acetic acid in 95% ethanol in a 1.5ml tube. Wipe the etched side of the shorter glass plate using a Kimwipes® tissue saturated with the freshly prepared binding solution. Be certain to wipe the entire plate surface with the saturated tissue. 5. Wait 5 minutes for the binding solution to dry. Wipe the shorter glass plate 3–4 times with 95% ethanol and Kimwipes® tissues to remove the excess binding solution. ! Failure to wipe excess binding solution from the shorter glass plate will cause the gel to stick to both plates, and the gel will be destroyed upon separation of the glass plates after electrophoresis. 6. Take special care not to allow the treated surfaces to touch each other. Assemble the glass plates by placing 0.4mm side spacers and a 0.4mm bottom spacer (optional) between the plates and using clamps to hold them in place. Lean the assembled plates against a test tube rack or other similar support. Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com Printed in USA. Revised 10/15 Part# TMD004 Page 13 5.B. Procedure (continued) 7. Prepare a 4% or 6% acrylamide solution (total of 75ml) by combining the ingredients listed below. Component urea deionized water 10X TBE buffer 40% acrylamide:bis (19:1) total volume 4% Gel 31.50g 40.00ml 3.75ml 7.50ml 75ml 6% Gel 31.50g 36.25ml 3.75ml 11.25ml 75ml Final Concentration 7M – 0.5X 4% or 6% Note: If preparing multiple gels on a daily basis, a larger 4% or 6% stock solution may be prepared, filtered as in Step 8 below, and stored at 4°C in the dark for up to one month. To prepare a single gel, remove 75ml of this stock solution, and continue with Step 9. 8. Filter the acrylamide solution through a 0.2 micron filter (e.g., Nalgene® tissue culture filter). 9. Pour the filtered acrylamide solution into a squeeze bottle. 10. Add 50µl of TEMED and 500µl of 10% ammonium persulfate to the acrylamide solution, and mix gently. 11. Carefully pour the acrylamide solution between the glass plates. To prevent bubble formation, start pouring at one side of the assembled plates and maintain a constant flow of solution. 12. Position the gel horizontally, resting it on two test tube racks or other similar supports. Remove any bubbles that may have formed. 13. Insert one or two 14cm doublefine (49 point) sharkstooth combs, straight side into the gel, between the glass plates (6mm of the comb should be between the two glass plates). If using a square-tooth comb, insert the comb between the glass plates until the teeth are almost completely inserted into the gel. 14. Secure the comb(s) with 2–3 clamps each. 15. Pour the remaining acrylamide solution into a disposable conical tube as a polymerization control. Rinse the squeeze bottle, including the spout, with water. 16. Allow polymerization to proceed for at least 1 hour. Check the polymerization control to be sure that polymerization has occurred. Note: The gel may be stored overnight if a paper towel saturated with deionized water and plastic wrap are placed around the well end of the gel to prevent the gel from drying out. If no bottom spacer is used, the bottom of the gel should be wrapped. Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com Part# TMD004 Page 14 Printed in USA. Revised 10/15 6. Polyacrylamide Gel Electrophoresis 6.A. Gel Pre-Run 1. Remove the clamps from the polymerized acrylamide gel, and clean the glass plates with paper towels saturated with deionized water. 2. Shave any excess polyacrylamide away from the comb. Remove the comb and bottom spacer. 3. Add 0.5X TBE to the bottom chamber of the electrophoresis apparatus. 4. Gently lower the gel and glass plates into the buffer with the longer plate facing out and the well-side on top. 5. Secure the glass plates to the sequencing gel apparatus. 6. Add 0.5X TBE to the top buffer chamber of the electrophoresis apparatus. 7. Using a 50–100cc syringe filled with buffer, remove the air bubbles on the top of the gel. Be certain the well area is devoid of air bubbles and small pieces of polyacrylamide. Use a syringe with a bent 19-gauge needle to remove the air bubbles between the glass plates on the bottom of the gel. 8. Pre-run the gel to achieve a gel surface temperature of approximately 50°C. Consult the manufacturer’s instruction manual for the recommended electrophoresis conditions. Note: As a reference, we generally use 60–65 watts for a 40cm polyacrylamide gel, 40–45 watts for a 32cm gel. The gel running conditions may have to be adjusted in order to reach a temperature of 50°C. 6.B. Sample Preparation 1. Prepare the PCR samples by mixing 2.5µl of each sample with 2.5µl of STR 2X Loading Solution. Note: The sample alleles may appear more intense than ladder alleles on the gel, but this should not interfere with allele determination. For more even band intensities, mix 1µl of each sample with 4µl of a premix containing 2.5µl of STR 2X Loading Solution and 1.5µl of STR 1X Buffer. 2. Add 2.5µl (50ng) of pGEM® DNA Markers to 2.5µl of STR 2X Loading Solution for each marker lane. Note: We recommend loading pGEM® DNA Markers into the first and last lanes of the gel. 3. Add 2.5µl of the Allelic Ladder Mix to 2.5µl of STR 2X Loading Solution for each allelic ladder lane. The number of allelic ladder lanes used depends on personal preference. Note: For combined CSF1PO, TPOX, TH01 multiplex and Amelogenin reactions, mix the corresponding allelic ladder mixes 1:1, then add 2.5µl of allelic ladder mix to 2.5µl of STR 2X Loading Solution. 4. Briefly centrifuge the samples in a microcentrifuge to bring the contents to the bottom of the tube. Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com Printed in USA. Revised 10/15 Part# TMD004 Page 15 6.C. Sample Loading 1. Denature the samples by heating at 95°C for 2 minutes, then immediately chill on crushed ice or in an ice-water bath. Note: Denature the samples just prior to loading the instrument. 2. After the pre-run (Section 6.A), use a 50–100cc syringe filled with buffer to flush the urea from the well area. If using a sharkstooth comb, carefully insert the comb teeth into the gel approximately 1–2mm. Leave the comb inserted in the gel during both gel loading and electrophoresis. 3. Load 3µl of each sample into the respective wells. The loading process should take no longer than 20 minutes to prevent the gel from cooling. Note: An organizational sheet for loading a gel is provided in Section 13.H. 6.D. Gel Electrophoresis 1. Once loading is complete, run the gel using the same conditions as in Section 6.A. If you are loading the gel multiple times, allow the gel to run 20–30 minutes before loading the next set of samples (Figure 4). This will prevent the samples from overlapping during electrophoresis. (Do not do this with multiplex systems.) Note: In a 6% gel, bromophenol blue migrates at approximately 25 bases and xylene cyanol migrates at approximately 105 bases. In a 4% gel, bromophenol blue migrates at approximately 40 bases and xylene cyanol migrates at approximately 170 bases. 2. Knowing the size ranges for each locus (Tables 7 and 8, Section 13.B) and migration characteristics of the dyes (Step 1, above), stop electrophoresis any time after the locus of interest has passed the midpoint of the gel. If running more than one locus or a multiplex, be careful not to run the smallest locus off the bottom of the gel. 3. Proceed to Section 7 for silver stain detection. Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com Part# TMD004 Page 16 Printed in USA. Revised 12/10 7. Silver Staining This protocol describes the use of the SILVER SEQUENCE™ Staining Reagents (Cat.# Q4132). One system contains sufficient reagents to stain 10 sequencing size gels and includes: • • • • • 500µl 20g 60ml 10ml 600g Bind Silane Silver Nitrate (10 × 2g) Formaldehyde, 37% (20 × 3ml) Sodium Thiosulfate, 10mg/ml (10 × 1ml) Sodium Carbonate (10 × 60g) Materials to Be Supplied by the User (Solution compositions are provided in Section 13.F.) • fix/stop solution • staining solution • developer solution (chilled to 4–10°C) • Nalgene® wash tubs (54.1 × 43.5 × 13cm or appropriate size for your system) • orbital shaker or rocker platform Use 2 liters of each solution per gel for each step (for a 54.1 × 43.5 × 13cm tray). 7.A. Procedure 1. After electrophoresis, empty the buffer chambers and carefully loosen the gel clamps. Remove the glass plates from the apparatus. 2. Place the gel and glass plates on a flat surface. Remove the comb and side spacers. Use a plastic wedge to carefully separate the two glass plates. The gel should be strongly affixed to the shorter glass plate. 3. Place the gel (attached to the shorter plate) in a shallow plastic tray (e.g., Nalgene® wash tub). 4. To silver stain, follow Steps a–h. Gently agitate during each step. ! Steps involving solutions containing formaldehyde should be performed in a chemical hood. Step a. b. c. d. e. f. Solution fix/stop solution (See Note 1) deionized water repeat Step b, twice staining solution deionized water (See Note 2) developer solution g. h. fix/stop solution (See Note 3) deionized water Time 20 minutes 2 minutes 2 × 2 minutes 30 minutes 10 seconds up to 5 minutes (until alleles and ladders are visible) 5 minutes 2 minutes Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com Printed in USA. Revised 12/10 Part# TMD004 Page 17 7.A. Procedure (continued) Notes: 5. 1. Save the fix/stop solution from Step 4a, to use in Step 4g. 2. The duration of Step 4e is important. The total time from immersion in deionized water to immersion in developer solution should be less than 20 seconds. If the deionized water rinse step does exceed 20 seconds, repeat Step 4d. 3. Add fix/stop solution directly to developer solution to stop developing reaction. Position the gel and shorter plate upright, and allow it to dry overnight. For best results, the gel should be completely dried before APC Film development. Alternatively, to create film prints of the gel immediately, cover the gel with plastic wrap, and proceed to Section 8. Figure 1, Section 10 shows typical results for the multiplex GenePrint ® STR Systems. Figure 3, Section 10, shows typical results for the Amelogenin locus and the STR loci, F13B, HPRTB and LPL using a dilution series of 250ng to 0.5ng of template DNA. 7.B. Reuse of Glass Plates 1. For disposal, immerse the plate and affixed gel in a 10% NaOH solution for 1 hour to overnight. Discard the gel, and clean the glass plate with deionized water and a detergent such as Liqui-Nox® detergent. The 10% NaOH solution may be reused for additional gels. 2. All cleaning utensils and sponges for the longer glass plates should be kept separate from those for the shorter glass plates to prevent crosscontamination of the binding solution. Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com Part# TMD004 Page 18 Printed in USA. Revised 10/15 8. Exposure of Film A direct image may be produced using Automatic Processor Compatible (APC) Film. The image produced on APC Film is the mirror image of the gel. Use of film allows the generation of multiple permanent images with more control over band and background intensity than does development of the gel alone. Handle all plates with gloved hands to avoid fingerprints. Materials to Be Supplied by the User (Solution compositions are provided in Section 13.F.) • white light box • automatic film processor or film developing tanks • Automatic Processor Compatible (APC) Film (Cat.# Q4411) 1. In the darkroom with a safelight on, place the dry, stained gel attached to the shorter plate (gel side up) on a white fluorescent light box. Note: For best results, the gel should be completely dry before the image is captured with APC film. If capturing an image from a gel that has not been dried, cover the gel with plastic wrap. 2. Position the APC Film, emulsion side down, over the gel to be copied. Note: The emulsion side of the film can be identified as the glossy white surface; the nonemulsion side has a gray tint. 3. Place a clean glass plate on top of the film to maintain contact between the gel and film. Turn on the white light box, and expose the film for 1–2 minutes, depending on the gel background level and the intensity of the white light. (This step must be optimized for individual light boxes.) 4. Develop the film as recommended by the manufacturer. APC film may be processed manually or with an automatic film processor. For automatic film processors, follow the manufacturer’s instructions. Note: The image produced on APC Film is the mirror image of the gel. 5. If there is very little signal, decrease the exposure time used in Step 3. If the film appears brown or black, increase the exposure time. Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com Printed in USA. Revised10/15 Part# TMD004 Page 19 9. Data Analysis For ease of interpretation, allelic ladders should be run in lanes adjacent to each sample. Direct comparison between the allelic ladders and amplified samples of the same locus allows for assignment of alleles. Note that microvariant alleles, such as the TH01 allele 9.3 and the F13A01 allele 3.2, do not co-migrate with allelic ladder fragments. In addition, mutations or rare alleles may be seen occasionally. The migration of such “off-ladder” alleles cannot be predicted. With silver stain detection, both DNA strands are detected. For some loci, such as TH01, FESFPS and vWA, the difference in the sequence of the opposing strands causes them to migrate at different rates. This results in doublets for each allele (Figure 1). This strand separation may be more pronounced with longer electrophoresis of gels as seen for the sequential loading of TH01 amplification products in Figure 4 (Section 10). Note that in the case of locus F13A01, more pronounced separation of opposing strands is observed with the larger alleles (see Figure 1). Artifact bands also may be detected with these systems. Shadow banding (16–18) or repeat slippage appears as faint bands one repeat unit (i.e., 4 bases) below the true alleles. This is most pronounced with the vWA locus (Figure 1). Terminal nucleotide addition occurs when Taq DNA polymerase catalyzes nontemplated addition of a nucleotide to the 3´-termini of amplified DNA fragments (18–20). A band that is one base shorter than the expected allele may result from the inefficiency of the terminal nucleotide addition. An artifact band is generated when this terminal addition does not occur with 100% efficiency. This may be visualized as an extra band, as seen in Figure 3 with the LPL and F13B loci. The addition of a final extension step (amplification protocols 7,8,9,10,11; Table 4) increases the amount of product that contains the added terminal nucleotide, thus minimizing the shorter artifact band (18). For the GenePrint ® SilverSTR® III System, performance variation of thermal cyclers may cause extraneous bands to be generated above the allele range. The use of AmpliTaq Gold® DNA polymerase with this system may minimize or eliminate these extra bands. Alternatively, raising the annealing temperature to 62°C can also minimize or eliminate these bands. At annealing temperatures higher than 62°C, the amplification of D7S820 alleles may be compromised. Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com Part# TMD004 Page 20 Printed in USA. Revised 10/15 9.A. pGEM® DNA Markers The pGEM® DNA Markers are visual standards used to confirm allelic size ranges for the loci. The markers consist of fifteen DNA fragments with the following sizes (in base pairs): 2,645 1,605 1,198 676 517 460 396 350 222 179 126 75 65 51 36 9.B Controls Observe the lanes containing the negative controls. They should be devoid of amplification products. Observe the lanes containing the positive K562 DNA positive controls. Compare the K562 DNA allelic repeat sizes with the locus-specific allelic ladder. The expected K562 DNA allele size(s) for each locus are listed in Tables 7 and 8, Section 13.B. 9.C. STR Ladders Each locus or multiplex has a characteristic allelic ladder. Please refer to Section 13.B for locus-specific allelic ladder information. In general, the allelic ladders contain fragments of the same lengths as either several or all known alleles for the locus. Visual comparison between the allelic ladder and amplified samples of the same locus allows precise assignment of alleles. Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com Printed in USA. Revised 10/15 Part# TMD004 Page 21 10. Representative STR Data CTT Multiplex FFv Multiplex L 1 2 L 3 4 L SilverSTR® III Multiplex L1 2L 3 4 L – 15 CSF1PO L – 16 F13A01 –7 1 2 L 3 4 L – 15 D16S539 –4 –5 – 14 – 13 FESFPS – 14 –7 D7S820 TPOX –6 –6 – 15 – 11 – 20 TH01 vWA – 13 –7 Figure 1. GenePrint ® STR Multiplex Systems. Individual genomic DNA samples (lanes 1–4) were amplified using GenePrint ® STR Systems as indicated and detected using silver staining as described in this manual. The amplification products using the CTT (CSF1PO, TPOX, TH01) Multiplex and the FFv (F13A01, FESFPS, vWA) Multiplex were separated using a 4% denaturing polyacrylamide gel. Amplification products using the SilverSTR® III (D16S539, D7S820, D13S317) System were separated in a 6% denaturing polyacrylamide gel. Lanes labeled (L) contain allelic ladders for the respective loci. Numbers to the right of each image indicate the smallest and largest number of repeat units present in corresponding fragments of each allelic ladder. Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com Part# TMD004 Page 22 Printed in USA. Revised 10/15 5808TA –5 D13S317 6% Denaturing Gel 4% Denaturing Gel L 1 2 L 3 4 L 5 6 L 1 L 15 2 L 3 4 L 5 6 L 15 D16S539 D16S539 5 5 14 14 D7S820 D7S820 6 6 D13S317 D13S317 7 5809TA 15 15 7 Figure 2. GenePrint ® SilverSTR® III System. Lanes 1–6 show amplification of 1ng of human DNA using the GenePrint ® SilverSTR® III System. Amplification products were separated using a 4% denaturing polyacrylamide gel and a 6% denaturing polyacrylamide gel and were detected by silver stain analysis. Lanes labeled L contain the SilverSTR® Allelic Ladder Mix. Numbers to the right of each image indicate the smallest and largest number of tandem repeat units present in corresponding fragments of each allelic ladder. Note that for the locus D16S539 the strands of the individual alleles separate forming doublets. This results from sequence differences between the two complementary strands, which affect their relative migration. In a 6% gel, the doublets are closely spaced and do not interfere with interpretation. In a 4% gel, however, these doublets separate such that the top strand from one allele overlaps with the bottom strand of the next larger allele, requiring greater care during interpretation. Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com Printed in USA. Revised 10/15 Part# TMD004 Page 23 A. B. 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 17 16 15 14 13 12 11 10 9 8 7 6 218bp (Y) 212bp (X) Amelogenin C. 1 2 3 4 5 6 7 8 HPRTB 14 13 D. 1 2 3 4 5 6 7 8 12 11 10 10 9 9 8 6 7 LPL F13B 0753TA08_4A 0753TA08_4A 7 Figure 3. Amplification of varying concentrations of K562 template DNA at different STR loci and the Amelogenin locus. K562 DNA was amplified at the Amelogenin locus and various STR loci. The Perkin-Elmer model 480 thermal cycler was used with protocols 1 or 2 (Table 4). The use of protocol 7 does not produce the lowest fragment of each trio seen in the F13B and LPL products (data not shown). For each panel, lanes 1 and 8 contain the locus-specific allelic ladder; lanes 2–6 contain amplified K562 DNA using 250, 25, 5, 1 and 0.5ng of starting template, respectively; lane 7 contains a negative control amplification reaction (i.e., no template DNA). Note: The F13B Allelic Ladder has been updated to include alleles 5 through 11. Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com Part# TMD004 Page 24 Printed in USA. Revised 10/15 1 L 2 3 L 4 5 L 6 7 L 8 0097TA07_3A 11 10 9 8 7 6 5 Load #3 Total time = 50 Minutes 11 10 9 8 7 6 5 Load #2 Total time = 80 Minutes 11 10 9 8 7 6 5 Load #1 Total time = 110 Minutes Figure 4. Sequential loading of STR locus TH01. For more efficient use of one 4% denaturing polyacrylamide gel, samples may be loaded at 30-minute intervals to obtain three times the amount of information when analyzing an individual STR system. Randomly selected DNA samples were amplified using the GenePrint ® STR System—TH01 as described in this manual. Samples and ladders were loaded at three different times: load #1 (time 0), load #2 (30 minutes after load #1) and load #3 (60 minutes after load #1). Following load #3, samples were run for an additional 50 minutes. Separated products were detected by silver stain analysis. Lanes L, TH01 Allelic Ladder; lanes 1–8, amplified DNA from several individuals. Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com Printed in USA. Revised 10/15 Part# TMD004 Page 25 11. Troubleshooting For questions not addressed here, please contact your local Promega Branch Office or Distributor. Contact information available at: www.promega.com. E-mail: [email protected] Symptoms Faint or no bands Bands are fuzzy throughout the lanes Causes and Comments Impure template DNA. Because of the small amount of template used, this is rarely a problem. Depending on the DNA extraction procedure used, inhibitors may exist in the DNA sample. Insufficient template DNA. Use the recommended amount of template DNA. Insufficient enzyme activity. Use the recommended amount of Taq DNA polymerase. Check the expiration date on the tube label. Wrong amplification program. Choose the correct amplification program for each locus. High salt concentration or altered pH. If the DNA template is stored in TE buffer that is not pH 8.0 or contains a higher EDTA concentration, the DNA volume should not exceed 20% of the total reaction volume. Carryover of K+, Na+, Mg2+ or EDTA from the DNA sample can negatively affect PCR. A change in pH may also affect PCR. Store DNA in TE–4 buffer (10mM Tris HCl [pH 8.0], 0.1mM EDTA) or nuclease-free water. Thermal cycler or tube problems. Review the thermal cycling protocols in Section 4. We have not tested other reaction tubes or thermal cyclers. Calibration of the thermal cycler heating block may be required. Primer concentration was too low. Use the recommended primer concentration. Mix well before use. Ice was not used to set up reactions. Set up the reactions on crushed ice. Very light allele intensity is obtained with some loci if ice is not used when setting up the reactions. The use of AmpliTaq Gold® DNA polymerase will also remedy this problem. Samples were not denatured before loading onto the gel. Be sure the samples are heated at 95°C for 2 minutes immediately prior to loading. Improper rinsing following staining. The rinse step was performed for more than 20 seconds. Longer rinses remove the silver deposited on the DNA. Rinse for a shorter time. Poor-quality water was used. Use ultrapure water (e.g., NANOpure®- or Milli-Q®-purified water) or double-distilled water. Poor-quality polyacrylamide gel. Prepare acrylamide and buffer solutions using high-quality reagents. Store acrylamide solutions in the dark. Electrophoresis temperature was too high. Run gel at lower temperature (40–60°C). Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com Part# TMD004 Page 26 Printed in USA. Revised 10/15 11. Troubleshooting (continued) Symptoms Background smearing within lanes Extra bands visible in one or all of the lanes Gel looks yellow after silver staining High background on gel Gel adheres to both plates Causes and Comments Poor-quality polyacrylamide gel. The amount of smearing may be reduced with high-quality polyacrylamide. Note: The allelic ladder for the D16S539 locus in the SilverSTR® III System shows some smearing. Sample overloading. Reduce the amount of template DNA used, or dilute the amplification reaction before mixing with loading buffer. BSA was used in amplification. The use of BSA is optional in the amplifications. BSA will stain with silver, resulting in a high-molecular-weight smear within the sample lane. Contamination with another template DNA or previously amplified DNA. Cross-contamination can be a problem. Use aerosol-resistant pipet tips, and change gloves regularly. Artifacts of STR amplification. PCR amplification sometimes generates artifacts that appear as faint bands one or four bases below an allele. Refer to Section 13.B for locus-specific information regarding this event. Artifacts of amplification with the GenePrint ® SilverSTR® III System. Performance variation of thermal cyclers may cause extraneous bands to be generated outside of the allele range. The use of AmpliTaq Gold® DNA polymerase with this system may minimize or eliminate these extra bands. Alternatively, raising the annealing temperature to 62°C can also minimize or eliminate these bands. At annealing temperatures higher than 62°C, the amplification of D7S820 alleles may be compromised. Samples were not completely denatured. Heat denature the samples at 95°C for 2 minutes immediately prior to loading the gel. Gel was left in the developer solution too long. Do not leave gel in developer solution for more than 5 minutes. Often, 2 minutes is sufficient. Excess silver nitrate was present. Rinse the gel for 10 seconds in deionized water before adding developer solution. Development was too long. Stop the development reaction after the appearance of the alleles and ladders. Longer glass plate was contaminated with binding solution, or treatment of the longer glass plate with Gel Slick® solution was inadequate. Wipe excessive binding solution from the short glass plate. Exercise care to avoid contaminating the longer glass plate with binding solution. Ensure uniform coverage of the longer plate with Gel Slick® solution. Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com Printed in USA. Revised 10/15 Part# TMD004 Page 27 12. References 1. Edwards, A. et al. (1991) DNA typing with trimeric and tetrameric tandem repeats: Polymorphic loci, detection systems, and population genetics. In: Proceedings from The Second International Symposium on Human Identification 1991, Promega Corporation, 31–52. 2. Edwards, A. et al. (1991) DNA typing and genetic mapping with trimeric and tetrameric tandem repeats. Am. J. Hum. Genet. 49, 74–56. 3. Edwards, A. et al. (1992) Genetic variation at five trimeric and tetrameric tandem repeat loci in four human population groups. Genomics 12, 241–53. 4. Warne, D. et al. (1991) Tetranucleotide repeat polymorphism at the human beta-actin related pseudogene 2 (ACTBP2) detected using the polymerase chain reaction. Nucl. Acids Res. 19, 6980. 5. Ausubel, F.M. et al. (1993) Unit 15: The polymerase chain reaction. In: Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley-Interscience, NY. 6. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Chapter 14: In vitro amplification of DNA by the polymerase chain reaction. In: Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 7. PCR Technology: Principles and Applications for DNA Amplification (1989) ed., Erlich, H.A., Stockton Press, NY. 8. PCR Protocols: A Guide to Methods and Applications (1990) eds., Innis, M.A. et al., Academic Press, San Diego, CA. 9. Puers, C. et al. (1993) Identification of repeat sequence heterogeneity at the polymorphic STR locus HUMTH01[AATG]n and reassignment of alleles in population analysis using a locus-specific allelic ladder. Am. J. Hum. Genet. 53, 953–8. 10. Hammond, H. et al. (1994) Evaluation of 13 short tandem repeat loci for use in personal identification applications. Am. J. Hum. Genet. 55, 175–89. 11. Bever, R.A. and Creacy, S. (1995) Validation and utilization of commercially available STR multiplexes for parentage analysis. In: Proceedings from the Fifth International Symposium on Human Identification 1994, Promega Corporation, 61–8. 12. Sprecher, C.J. et al. (1996) General approach to analysis of polymorphic short tandem repeat loci. BioTechniques 20, 266–76. 13. Lins, A.M. et al. (1996) Multiplex sets for the amplification of polymorphic short tandem repeat loci— silver stain and fluorescent detection. BioTechniques 20, 882–9. 14. Presley, L.A. et al. (1992) The implementation of the polymerase chain reaction (PCR) HLA DQ alpha typing by the FBI laboratory. In: Proceedings from the Third International Symposium on Human Identification 1992, Promega Corporation, 245–69. 15. Hartmann, J.M. et al. (1991) Guidelines for a quality assurance program for DNA analysis. Crime Laboratory Digest 18, 44–75. 16. Levinson, G. and Gutman, G.A. (1987) Slipped-strand mispairing: A major mechanism for DNA sequence evolution. Mol. Biol. Evol. 4, 203–21. 17. Schlotterer, C. and Tautz, D. (1992) Slippage synthesis of simple sequence DNA. Nucl. Acids Res. 20, 211–5. 18. Walsh, P.S., Fildes, N.J. and Reynolds, R. (1996) Sequence analysis and characterization of stutter products at the tetranucleotide repeat locus vWA. Nucl. Acids Res. 24, 2807–12. 19. Smith, J.R. et al. (1995) Approach to genotyping errors caused by nontemplated nucleotide addition by Taq DNA polymerase. Genome Res. 5, 312–7. 20. Magnuson, V.L. et al. (1996) Substrate nucleotide-determined non-templated addition of adenine by Taq DNA polymerase: Implications for PCR-based genotyping. BioTechniques 21, 700–9. Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com Part# TMD004 Page 28 Printed in USA. Revised 10/15 12. References (continued) 21. Bassam, B.J., Caetano-Anolles, G. and Gresshoff, P.M. (1991) Fast and sensitive silver staining of DNA in polyacrylamide gels. Anal. Biochem. 196, 80–3. 22. Budowle, B. et al. (1991) Analysis of the VNTR locus D1S80 by the PCR followed by high-resolution PAGE. Am. J. Hum. Genet. 48, 137–44. 23. Nakamura, Y. et al. (1987) Variable number of tandem repeat (VNTR) markers for human gene mapping. Science 235, 1616–22. 24. Budowle, B. and Monson, K.L. (1989) In: Proceedings of an International Symposium on the Forensic Aspects of DNA Analysis, Government Printing Office, Washington, D.C. 25. Bär, W. et al. (1997) DNA Recommendations: Further report of the DNA Commission of the ISFH regarding the use of short tandem repeat systems. Int. J. Leg. Med. 110, 175–6. 26. Puers, C. et al. (1994) Analysis of polymorphic STR loci using well-characterized allelic ladders. In: Proceedings from the Fourth International Symposium on Human Identification 1993, Promega Corporation, 161–72. 27. Puers, C. et al. (1994) Allelic ladder characterization of the short tandem repeat polymorphism located in the 5´ flanking region to the human coagulation factor XIII A subunit gene. Genomics 23, 260–4. 28. Jones, D.A. (1972). Blood samples: Probability of discrimination. J. Forensic Sci. Soc. 12, 355–9. 29. Brenner, C. and Morris, J.W. (1990) In: Proceedings from the International Symposium on Human Identification 1989, Promega Corporation, 21–53. 30. Mandrekar, P.V., Krenke, B.E. and Tereba, A. (2001) DNA IQ™: The intelligent way to purify DNA. Profiles in DNA 4(3), 16. 31. Mandrekar, M.N. et al. (2001) Development of a human DNA quantitation system. Profiles in DNA 4(3), 9–12. 32. Greenspoon, S. and Ban, J. (2002) Robotic extraction of sexual assault samples using the Biomek® 2000 and the DNA IQ™ System. Profiles in DNA 5(1), 3–5. 33. Comey, C. et al. (1994) DNA extraction strategies for amplified fragment length polymorphism analysis. J. Forensic Sci. 39, 1254–69. 34. Lins, A. et al. (1998) Development and population study of an eight-locus short tandem repeat (STR) multiplex system. J. Forensic Sci. 43, 1168–80. Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com Printed in USA. Revised 10/15 Part# TMD004 Page 29 13. Appendix 13.A.Advantages of STR Typing The GenePrint ® STR Systems provide a rapid, non-radioactive method, which can be used to evaluate very small amounts (e.g., 1ng) of human DNA. The protocols detailed in this manual describe the use of silver staining (21) to detect the presence of amplified STR products following their separation by denaturing polyacrylamide gel electrophoresis. Information on detecting STR products by fluorescence methods is available at: www.promega.com STR typing is more tolerant of the use of degraded DNA templates than other methods of individual identification because the amplification products are less than 400bp long, much smaller than the material detected with AMP-FLP (22) or VNTR (23) analysis. This format is also amenable to a variety of rapid DNA purification techniques. In addition to these advantages, the STR loci chosen for inclusion in the GenePrint ® Systems contain alleles of discrete and separable lengths. This allows the construction of allelic ladders, which contain fragments of the same lengths as several or all known alleles for the locus. Visual comparison between the allelic ladder and amplified samples of the same locus allows rapid and precise assignment of alleles. Results obtained using the GenePrint ® STR Systems can be recorded in a digitized format, allowing direct comparison with stored databases. Population analyses do not require the use of arbitrarily defined fixed bins for population data (24). 13.B. Advantages of Using the Loci in the GenePrint ® STR Systems The STR loci and primers contained in the GenePrint ® STR Systems (Tables 6 and 7) have been carefully selected to minimize artifacts, including those associated with Taq DNA polymerase, such as repeat slippage and terminal nucleotide addition as well as genetic artifacts called microvariant alleles. Repeat slippage (16–18), sometimes called “n–4 bands”, “stutter” or “shadow bands”, is due to the loss of a repeat unit during DNA amplification. The amount of this artifact observed depends primarily on the locus and the DNA sequence being replicated. We have chosen loci that exhibit little or no repeat slippage. The vWA locus is an exception, revealing as much as 10% stutter. This locus has been included primarily for its popularity in the forensic DNA-testing community. Terminal nucleotide addition occurs when Taq DNA polymerase adds a nucleotide, generally adenine, to the ends of amplified DNA fragments in a template-independent manner (19,20). The efficiency with which this occurs varies with different primer sequences. Thus, an artifact band one base shorter than expected (i.e., missing the terminal addition) is sometimes seen. Redefinition of the primer sequences and/or the addition of a final extension step of 60°C for 30 minutes to the amplification protocol can lead to essentially full terminal nucleotide addition (18). Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com Part# TMD004 Page 30 Printed in USA. Revised 10/15 Table 6. Locus-Specific Information. STR Locus Amelogenin1 Chromosomal Location Xp22.1–22.3 and Y GenBank® Locus and Locus Definition HUMAMEL, Human Y chromosomal gene for amelogenin-like protein Repeat Sequence 5´→ 3´ NA CSF1PO 5q33.3–34 HUMCSF1PO, Human c-fms proto-oncogene for CSF-1 receptor gene AGAT2 D16S539 16q24–qter NA AGAT2 D7S820 7q11.21–22 NA AGAT2 D13S317 13q22–q31 NA AGAT2 F13A01 6p24.3–p25.1 HUMF13A01, Human coagulation factor XIII a subunit gene AAAG2 F13B 1q31–q32.1 AAAT2 FESFPS 15q25–qter HPRTB Xq26 HUMBFXIII, Human factor XIII b subunit gene HUMFESFPS, Human c-fes/fps proto-oncogene HUMHPRTB, Human hypoxanthine phosphoribosyltransferase gene LPL 8p22 AAAT2 TH01 11p15.5 TPOX 2p25.1–pter vWA (formerly vWF) 12p12–pter HUMLIPOL, Human lipoprotein lipase gene HUMTH01, Human tyrosine hydroxylase gene HUMTPOX, Human thyroid peroxidase gene HUMVWFA31, Human von Willebrand factor gene AAAT2 AGAT2 AATG2 AATG2 AGAT2 NA = not applicable. 1Amelogenin is not an STR, but displays a 212-base, X-specific band and a 218-base, Y-specific band. K562 DNA (female) displays only the 212-base, X-specific band. 2Repeat sequences represent all four possible permutations (e.g., AGAT is used for AGAT, GATA, ATAG or TAGA). The first alphabetic representation of the repeat (e.g., AGAT) is used according to the precedent of Edwards et al. (2). The published article, “DNA Guidelines: Further Report of the DNA Commission of the ISFH Regarding the use of Short Tandem Repeat Systems” (25) describes different rules for STR allele nomenclature. Allele designations for all listed loci are identical using both methods except for the locus F13B. In this case, alleles are one repeat unit larger when using the method described by the ISFH. For this locus, the community will have to decide whether to follow the new nomenclature or maintain the Edwards nomenclature to avoid confusion. The DNA Commission of the ISFH states “If a repeat designation of a commonly used STR system does not follow these guidelines, the established nomenclature for the sequence can continue to be used to avoid new confusion”. Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com Printed in USA. Revised 10/15 Part# TMD004 Page 31 Table 7. Additional Locus-Specific Information. Allelic Ladder Size Range1 (bases) 212–218 STR Ladder Alleles (# of repeats)2 NA CSF1PO 295–327 D16S539 264–304 D7S820 215–247 D13S317 165–197 F13A01 283–331 F13B 169–189 7,8,9,10,11, 12,13,14,15 5,8,9,10,11, 12,13,14,15 6,7,8,9,10, 11,12,13,14 7,8,9,10,11, 12,13,14,15 4,5,6,7,8,9,11, 12,13,14,15,16 6,7,8,9,10,11 FESFPS 222–250 HPRTB 259–303 LPL STR Locus Amelogenin4 Other Known K562 DNA Alleles3 Allele Sizes (# of repeats) (# of repeats) None 212,212 Comments 1,2 6 10,9 1 None 12,11 1 None 11,9 1 None 8,8 1 3.2,105 5,46 1,3 12 10,10 1 7,8,9,10,11,12,13,14 None 12,10 1 None 13,13 1 105–133 6,7,8,9,10,11, 12,13,14,15,16,17 7,9,10,11,12,13,14 8 12,10 1 TH01 179–203 5,6,7,8,9,10,11 9.3 9.3,9.3 1,4 TPOX 224–252 6,7,8,9,10,11,12,13 None 9,8 1 vWA (formerly vWF) 139–167 13,14,15,16, 17,18,19,20 11,21 16,16 1 NA = not applicable. 1Lengths of each allele in the allelic ladders have been confirmed by sequence analyses. 2Alleles in bold are present in greater amounts than other alleles. This simplifies interpretation. 3Alleles that represent <0.2% of the population may not be listed in this table. 4Amelogenin is not an STR, but displays a 212 base X-specific band and a 218 base Y-specific band. K562 DNA (female) displays only the 212 base X-specific band. 5Allele 10 (307 bases) is not included because it is rare and its exclusion creates a gap that simplifies interpretation of the allelic ladder (26,27). 6F13A01 allele 5 appears more intense than allele 4 in the K562 control sample. The K562 strain contains an unusual number of chromosomes, and some are represented more than twice per cell. It is hypothesized that in this strain the allele 5 version of chromosome 6 is present twice, while the allele 4 version of chromosome 6 is present only once. Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com Part# TMD004 Page 32 Printed in USA. Revised 12/10 Table 8. Multiplex System Information. Allelic STR Ladder GenePrint ® STR Component Ladder Size Alleles (number Range (bases) of repeats) Multiplex Cat.# Loci 2 CSF1PO 295–327 7,8,9,10,11, “CTT triplex” 12,13,14,15 (DC6000 and TPOX 224–252 6,7,8,9,10,11,12,13 DC6001) TH01 179–203 5,6,7,8,9,10,11 “FFv triplex” F13A01 283–331 (DC6030 and DC6031) FESFPS 222–250 vWA 139–167 “Silver-STR® III triplex” D16S539 264–304 D7S820 215–247 D13S317 165–197 (DC6450 and DC6451) 1Alleles Other K562 DNA Known Allele Alleles1 Sizes Comments 6 10,9 1,5 None 9,8 1,5 9.3 9.3,9.3 1,4,5 4,5,6,7,8,9,10, 11,12,13,14,15,16 7,8,9,10,11,12,13,14 3.2,10 53,4 1,3,5 None 12,10 1,5 13,14,15,16, 17,18,19,20 5,8,9,10,11, 12,13,14,15 6,7,8,9,10, 11,12,13,14, 7,8,9,10,11, 12,13,14,15 11,21 16,16 1,5 None 12,11 1,5 None 11,9 1,5 None 8,8 1,5 that represent <0.2% of the population may not be listed in this table. 2The GenePrint ® Sex Identification System—Amelogenin primers may be combined with the CTT primers to allow simultaneous amplification of all four loci as described in Section 4.B. The results provide information regarding the gender of the individual who contributed the DNA sample, as well as the STR information. Ordering information for the Amelogenin system may be found in Section 13.I. 3F13A01 allele 5 appears more intense than allele 4 in the K562 control sample. The K562 strain is known to contain an unusual number of chromosomes, and some are represented more than twice per cell. It is hypothesized that in this strain the allele 5 version of chromosome 6 is present twice while the allele 4 version of chromosome 6 is present only once. Comments on Tables 7 and 8 1. PCR amplification sometimes generates artifacts that appear as faint bands below the alleles. These products probably result from a process known as slippage, commonly observed in PCR amplification of regions that contain tandem repeats of short sequences (16–18). This characteristic is most pronounced with the vWA locus. 2. A strong extra band may be observed below the 212bp Amelogenin allele when more than 25ng of template DNA is amplified. 3. Locus F13A01 has a common allele 3.2. It contains 4 copies of the repeat but has a 2-base deletion in the region flanking the repeat (26,27). 4. Locus TH01 has a common 9.3 allele (9). A one-base deletion is present in the allele that contains 10 repeats. Note that reference 9 refers to this allele as 10–1 rather than 9.3. This allele was renamed 9.3 at the ISFH Conference in Venice, Italy, in October of 1993. 5. A background haze of silver stain is sometimes seen in the region in and above the allelic ladder. Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com Printed in USA. Revised 12/10 Part# TMD004 Page 33 13.C. Power of Discrimination Table 9 shows the matching probability (28) for the multiplex GenePrint ® STR Systems in various populations. When taken together, the triplexes described in this manual produce matching probabilities ranging from 1 in 1,030,000,000 in Caucasian-Americans to 1 in 5,180,000,000 in African-Americans. A measure of discrimination often used in paternity analyses is the paternity index (PI), a means for presenting the genetic odds in favor of paternity given the genotypes for the mother, child and a tested man (29). The typical PIs for the multiplex GenePrint ® STR Systems are shown in Table 10. The three triplexes together give typical paternity indices exceeding 500 in each group, enough to satisfy routine requirements for paternity determination. An alternative calculation used in paternity analyses is the power of exclusion (29). This value, calculated for the combined triplexes, exceeds 0.9985 in all populations tested (Table 11). Table 9. Matching Probability of Various Populations. Matching Probability STR System CTT triplex (CSF1PO, TPOX, TH01) FFv triplex (F13A01, FESFPS, vWA) African-American Caucasian-American Hispanic-American 1 in 1,590 1 in 435 1 in 549 1 in 2,828 1 in 927 1 in 1,343 1 in 1,152 1 in 2,552 1 in 2,493 1 in 5.18 × 109 1 in 1.03 × 109 1 in 1.84 × 109 SilverSTR® III triplex (D16S539, D7S820, D13S317) All 3 triplexes (9 loci) Table 10. Typical Paternity Indices of the Multiplex GenePrint ® STR Systems in Various Populations. Typical Paternity Index STR System African-American CTT triplex (CSF1PO, TPOX, TH01) 10.2 FFv triplex (F13A01, FESFPS, vWA) 16.0 Caucasian-American Hispanic-American 6.8 5.2 9.8 7.8 7.6 7.7 14.1 1233 521 563 SilverSTR® III triplex (D16S539, D7S820, D13S317) All 3 triplexes (9 loci) Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com Part# TMD004 Page 34 Printed in USA. Revised 10/15 Table 11. Power of Exclusion of the GenePrint ® STR Systems in Various Populations. Power of Exclusion STR System African-American CTT triplex (CSF1PO, TPOX, TH01) 0.906 FFv triplex (F13A01, FESFPS, vWA) 0.938 SilverSTR® III triplex (D16S539, D7S820, D13S317) All 3 triplexes (9 loci) Caucasian-American Hispanic-American 0.869 0.830 0.904 0.881 0.877 0.880 0.929 0.9993 0.9985 0.9986 13.D. DNA Extraction and Quantitation Methods The DNA IQ™ System (Cat.# DC6700) is a DNA isolation and quantitation system designed specifically for forensic and paternity samples (30). This novel system uses paramagnetic particles to prepare clean samples for STR analysis easily and efficiently and can be used to extract DNA from stains or liquid samples, such as blood or solutions. The DNA IQ™ Resin eliminates PCR inhibitors and contaminants frequently encountered in casework samples. With larger samples, the DNA IQ™ System delivers a consistent amount of total DNA. The system has been used to isolate and quantify DNA from routine sample types including buccal swabs, stains on FTA® paper and liquid blood. Additionally, DNA has been isolated from casework samples such as tissue, differentially separated sexual assault samples and stains on support materials. For applications requiring human-specific DNA quantification, the AluQuant® Human DNA Quantitation System (Cat.# DC1010) has been developed (31). The DNA IQ™ System and AluQuant® Human DNA Quantitation System have been fully automated on the Beckman Coulter Biomek® 2000 Laboratory Automation Workstation (32). For information about automation of laboratory processes on Beckman Coulter or other workstations, contact your local Promega Branch Office or Distributor (contact information available at: www.promega.com/worldwide/) or e-mail: [email protected] Note: For stains from blood and saliva, scientists at the FBI Academy have suggested an alternative method for DNA extraction (see reference 33). Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com Printed in USA. Revised 10/15 Part# TMD004 Page 35 13.E. Agarose Gel Electrophoresis of Amplification Products (Optional) This procedure is optional if PCR is routinely performed in your laboratory. Agarose gel electrophoresis can be used to rapidly confirm the success of the amplification reaction prior to performing polyacrylamide gel electrophoresis. Materials to Be Supplied by the User (Solution compositions are provided in Section 13.F.) • TAE 1X buffer • agarose • 5X loading solution • ethidium bromide solution, 0.5µg/ml ! Ethidium bromide is a powerful mutagen. Wear gloves at all times, and use a mask when weighing out ethidium bromide powder. 1. Prepare a 2% agarose gel (approximately 150cm2) by adding 2.0g of agarose to 100ml of TAE 1X buffer. Mark the liquid level on the container, then boil or heat in a microwave oven to dissolve the agarose. Add preheated (60°C) deionized water to make up for any volume lost due to evaporation. 2. Cool the agarose to 55°C before pouring into the gel tray. Be sure that the gel tray is level. Pour the agarose into the tray, insert the gel comb, and allow to set for 20–30 minutes. 3. Prepare the samples by mixing 10µl of each amplified sample with 2.5µl of 5X loading solution. 4. Prepare 1 liter of TAE 1X buffer for the electrophoresis running buffer. 5. Place the gel and tray in the electrophoresis gel box. Pour enough running buffer into the tank to cover the gel to a depth of at least 0.65cm. Gently remove the comb. 6. Load each sample mixed with 5X loading solution (prepared in Step 3). 7. Set the voltage at 5 volts/cm (measured as the distance between the two electrodes). Allow the gel to run for 2 hours. 8. After electrophoresis, stain the gel in TAE 1X buffer containing 0.5µg/ml ethidium bromide. Gently rock for 20 minutes at room temperature. Remove the ethidium bromide solution, and replace with deionized water. Allow the gel to destain for 20 minutes. 9. Using a UV transilluminator (302nm), photograph the gel (e.g., with Polaroid® 667 film). Note: When analyzing the data, do not be alarmed by extra bands in addition to the alleles. DNA heteroduplexes can be expected when performing nondenaturing agarose gel electrophoresis. The sole purpose of the agarose gel is to confirm the success of the PCR reaction. Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com Part# TMD004 Page 36 Printed in USA. Revised 10/15 13.F. Composition of Buffers and Solutions 0.5% acetic acid in 95% ethanol ethidium bromide stock solution Add 1ml of glacial acetic acid to 199ml of 95% ethanol. Add 1g of ethidium bromide to 100ml of deionized water. Stir on a magnetic stirrer until dye is dissolved. Wrap the container in aluminum foil, or transfer to a dark bottle. Store at room temperature. 40% acrylamide:bis (19:1) Dissolve 380g of acrylamide and 20g of bisacrylamide in 500ml of deionized water. Bring volume to 1 liter with deionized water. 10% ammonium persulfate GoldST*R 10X Buffer 500mM 100mM KCl Tris-HCl (pH 8.3 at 25°C) MgCl2 Triton® X-100 each dNTP BSA Add 0.5g of ammonium persulfate to 5ml of deionized water. Use 500µl for one acrylamide gel solution (75ml). Store the remaining volume in 500µl aliquots at –20°C. 15mM 1% 2mM 1.6mg/ml developer solution 5X loading solution 3ml 400µl 2L 60g 37% formaldehyde (H2CO) 10mg/ml sodium thiosulfate (Na2S2O3 • 5H2O) deionized water sodium carbonate (Na2CO3) 5% 0.1% 0.1% 100mM 10mM Ficoll® 400 bromophenol blue xylene cyanol EDTA (Na2EDTA • 2H2O) Tris-HCl (pH 7.5) fix/stop solution (10% acetic acid) 200ml 1,800ml glacial acetic acid deionized water Sodium carbonate must be ACS grade. We have confirmed quality with material from Fisher Scientific (Fisher Scientific Cat.# S263-500). Results may vary depending on source. sodium thiosulfate solution (10mg/ml) Prepare the solution, and chill to 10°C before use. Use only highquality deionized water and sodium carbonate. Prepare fresh before each use. staining solution 0.5M EDTA (pH 8.0) stock 186.1g Na2EDTA • 2H2O Add EDTA to 800ml of deionized water with vigorous stirring. Adjust the pH to 8.0 with NaOH (about 20g of NaOH pellets). Adjust the final volume to 1 liter. Dispense into aliquots, and sterilize by autoclaving. Add 5g of sodium thiosulfate (Na2S2O3 • 5H2O) to 500ml of deionized water. 2g 3ml 2,000ml silver nitrate (AgNO3) 37% formaldehyde (H2CO) deionized water STR 2X Loading Solution 10mM 95% 0.05% 0.05% NaOH formamide bromophenol blue xylene cyanol FF Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com Printed in USA. Revised 10/15 Part# TMD004 Page 37 13.F. Composition of Buffers and Solutions (continued) STR 10X Buffer 500mM 100mM 15mM 1% 2mM KCl Tris-HCl (pH 9.0) at 25°C MgCl2 Triton® X-100 each dNTP 50X TAE buffer (pH 7.2) 242g 57.1ml 100ml Tris base glacial acetic acid 0.5M EDTA stock Add the Tris base and EDTA stock to 500ml of deionized water. Add the glacial acetic acid. Bring to 1 liter with deionized water. 0.5X TBE buffer Add 50ml of 10X TBE to 950ml of deionized water. 10X TBE buffer 107.8g 7.44g ~55.0g Tris base EDTA (Na2EDTA • 2H2O) boric acid Dissolve the Tris base and EDTA in 800ml of deionized water. Add slightly less than the total amount of boric acid. Mix until completely dissolved, check the pH, and adjust to 8.3 with boric acid. Bring the volume to 1 liter with deionized water. TE–4 buffer (10mM Tris-HCl, 0.1mM EDTA [pH 7.5]) 1.21g 0.037g 2H2O) Tris base EDTA (Na2EDTA • Dissolve the Tris base and EDTA in 900ml of deionized water. Adjust to pH 7.5 with HCl. Increase volume to 1 liter with deionized water. 13.G. Population Data Allele frequencies for African-Americans, Caucasian-Americans and HispanicAmericans were generated as part of a collaborative effort between Genetic Design, Inc. (Greensboro, NC), and Promega Corporation (34). This population data can be found at: www.promega.com/techserv/apps/hmnid/ referenceinformation/popstat/custstat_Allelefreq.htm Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com Part# TMD004 Page 38 Printed in USA. Revised 10/15 13.H. Organizational Sheets Sample Preparation Tube Number Sample ID negative control Sample Conc. (ng/µl) – Sample (µl)/reaction 0 Sterile Water (µl) 2.5 Tube Number Sample ID Sample Conc. (ng/µl) Sample (µl)/reaction Sterile Water (µl) Tube Number Sample ID Sample Conc. (ng/µl) Sample (µl)/reaction Sterile Water (µl) Tube Number Sample ID Sample Conc. (ng/µl) Sample (µl)/reaction Sterile Water (µl) Tube Number Sample ID Sample Conc. (ng/µl) Sample (µl)/reaction Sterile Water (µl) Tube Number Sample ID Sample Conc. (ng/µl) Sample (µl)/reaction Sterile Water (µl) Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com Printed in USA. Revised 10/15 Part# TMD004 Page 39 Master Mix Preparation Date: Name: GenePrint ® STR Systems Locus = Reaction volume (sample + master mix) = Number of reactions = Master Mix Component Lot Number 25µl Volume Per × Sample (µl) 17.45 for monoplex × 17.35 for quadriplex sterile water = Final Volume (µl) = 2.50 × = 2.50 0.05 for monoplex 0.15 for quadriplex × = × = STR 10X Buffer1 10X Primer Pair Taq DNA polymerase (5u/µl)2 Number of Reactions total volume = 1If using AmpliTaq Gold® DNA polymerase, use the GoldST*R 10X Buffer (Cat.# DM2411, available separately) instead of the STR 10X Buffer. 2The volume given assumes a Taq DNA polymerase concentration of 5u/µl. For different enzyme concentrations, the volume of enzyme added must be adjusted accordingly. To assemble reactions, add 2.5µl DNA to each tube containing 22.5µl of master mix. Thermal Cycling Profile Perkin-Elmer Thermal Cycler Model Number: Annealing Temperature: File Number: Full Program Description: ______cycles: _______°C _______ minutes _______°C _______ minutes _______°C _______ minutes ______cycles: _______°C _______ minutes _______°C _______ minutes _______°C _______ minutes Hold: 4°C indefinitely Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com Part# TMD004 Page 40 Printed in USA. Revised 10/15 Experiment Date: Name: Electrophoresis Pre-run: minutes Starting time: Stopping time: Watts: Watts: Milliamps: Milliamps: Voltage: Voltage: Notes Gel Number: Lane Sample # Description Lane 1 1 2 2 3 3 4 4 5 5 6 6 7 7 8 8 9 9 10 10 11 11 12 12 13 13 14 14 15 15 16 16 17 17 18 18 19 19 20 20 21 21 22 22 23 23 24 24 25 25 Sample # Description Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com Printed in USA. Revised 10/15 Part# TMD004 Page 41 13.I. Related Products Fluorescent STR Multiplex Systems Product PowerPlex® 16 System GenePrint® GammaSTR® Multiplex (Fluorescein) D16S539, D7S820, D13S317, D5S818 GenePrint® Fluorescent STR CSF1PO, TPOX, TH01, vWA Multiplex (Fluorescein) Size 100 reactions 400 reactions Cat.# DC6531 DC6530 100 reactions DC6071 100 reactions DC6301 Size 1.2ml 12ml 50ml (2 × 25ml) Cat.# DM2411 DY1151 P1193 Size 100 reactions 400 reactions 50 samples 200 samples 10 pack Cat.# DC6701 DC6700 DC6801 DC6800 V1391 Size 25g 1L 1kg 3ml Cat.# V3131 V4251 V3171 DV4351 Not for Medical Diagnostic Use. Accessory Components Product GoldST*R 10X Buffer Mineral Oil Nuclease-Free Water Sample Preparation Systems Product DNA IQ™ System Differex™ System* Slicprep™ 96 Device *Not for Medical Diagnostic Use. Polyacrylamide Gel Electrophoresis Reagents Product Ammonium Persulfate TBE Buffer, 10X Urea Blue Dextran Loading Solution 14. Summary of Changes The 10/15 version of this document has been updated to remove expired patent and disclaimer statements and update the related products listing. Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com Part# TMD004 Page 42 Printed in USA. Revised 10/15 (a).Australian Pat. No. 724531, Korean Pat. No. 290332, Singapore Pat. No. 57050, Japanese Pat. No. 3602142 and other patents pending. © 2010–2015 Promega Corporation. All Rights Reserved. AluQuant, GammaSTR, GenePrint, pGEM, PowerPlex and SilverSTR are registered trademarks of Promega Corporation. Differex, DNA IQ and Slicprep are trademarks of Promega Corporation. AmpliTaq, AmpliTaq Gold and GeneAmp are registered trademarks of Roche Molecular Systems, Inc. Biomek is a registered trademark of Beckman Coulter, Inc. Costar is a registered trademark of Corning, Inc. Ficoll is a registered trademark of GE Healthcare Bio-sciences. FTA is a registered trademark of Flinders Technologies, Pty, Ltd., and is licensed to Whatman. Gel Slick is a registered trademark of BioWhittaker. GenBank is a registered trademark of the U.S. Dept. of Health and Human Services. Kimwipes is a registered trademark of Kimberly-Clark. Liqui-Nox is a registered trademark of Alconox. MicroAmp is a registered trademark of Applera Corporation. Milli-Q is a registered trademark of Millipore Corporation. Nalgene is a registered trademark of Nalge Nunc International. NANOpure is a registered trademark of Barnstead/Thermolyne Corporation. Nonidet is a registered trademark of Shell International Petroleum Company, Ltd. Parafilm is a registered trademark of American National Can Company. Polaroid is a registered trademark of Polaroid Corporation. Spin-X is a registered trademark of Costar Corporation. Triton is a registered trademark of Union Carbide Chemicals and Plastics Technology Corporation. Tween is a registered trademark of ICI Americas, Inc. Products may be covered by pending or issued patents or may have certain limitations. Please visit our Web site for more information. All prices and specifications are subject to change without prior notice. Product claims are subject to change. Please contact Promega Technical Services or access the Promega online catalog for the most up-to-date information on Promega products. Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com Printed in USA. Revised 10/15 Part# TMD004 Page 43
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