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1. 33 9 F Composition of Buffers and Solutions 34 9 G Related Products 35 9 H Summary of Changes 36 2 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD016 Revised 7 15 www promega com 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 may be detected using the polymerase chain reaction 5 8 Alleles of STR loci are differentiated by the number of copies of the repeat sequence contained within the amplified region and are distinguished from one another using fluorescence detection following electrophoretic separation The PowerPlex 16 BIO System a e is used for human identification applications including forensic analysis relation ship testing and research use The system allows coamplification and three color detection of sixteen loci fifteen STR loci and Amelogenin The PowerPlex 16 BIO
2. microcentrifuge 0 5ml or 0 2ml thin walled microcentrifuge tubes MicroAmp optical 96 well reaction plate or MicroAmp 8 strip reaction tubes Applied Biosystems aerosol resistant pipette tips see Section 9 G AmpliTaq Gold DNA polymerase Applied Biosystems Nuclease Free Water Cat P1193 Mineral Oil Cat DY1151 for use with the thermal cycler model 480 Note If using the GeneAmp PCR system 9600 9700 or 2400 thermal cyclers use 0 2ml MicroAmp 8 strip reaction tubes or MicroAmp plate For the Perkin Elmer model 480 we recommend 0 5ml GeneAmp thin walled reaction tubes We routinely amplify 0 5 1ng of template DNA in a 25 l reaction volume using the protocols detailed below Expect to see more intense bands for smaller loci and less intense bands for larger loci if more than the recommended amount of template is used Reduce the amount of template DNA or the number of cycles to correct this The PowerPlex 16 BIO System is optimized for the GeneAmp PCR system 9600 thermal cycler Amplification protocols for the GeneAmp PCR systems 9700 and 2400 thermal cyclers and Perkin Elmer model 480 thermal cycler are provided 4 A Amplification Setup The use of gloves and aerosol resistant pipette tips is highly recommended to prevent cross contamination Keep all pre amplification and post amplification reagents in separate rooms Prepare amplification reactions in a room dedicated for reacti
3. Rhodamine Red X FGA D8S1179 TPOX vWA Amel B L 1 2 3 4 5 577nm Scan JOE Penta D D16S539 CSF1PO D7S820 D13S317 D5S818 A L 1 2 3 4 5 505nm Scan Fluorescein Penta E D18S51 D21S11 TH01 D3S1358 Figure 4 Amplification of various template amounts with the PowerPlex 16 BIO System A single source DNA template 2 1 0 5 or 0 2ng was amplified The results are shown in lanes 1 4 respectively Lane 5 shows the amplification results with no DNA template Lanes labeled L contain the PowerPlex 16 BIO Allelic Ladder All materials were separated using a 6 PAGE PLUS acrylamide gel and detected using the Hitachi FMBIO II Fluorescence Imaging System Panel A A scan using a 505nm filter showing the fluorescein labeled loci Penta E D18S51 D21S11 TH01 and D3S1358 Panel B A scan using a 577nm filter showing the JOE labeled loci Penta D CSF1PO D16S539 D7S820 D13S317 and D5S818 Panel C A scan using a 598nm filter showing the Rhodamine Red X labeled loci FGA TPOX D8S1179 vWA and Amelogenin Panel D A scan using a 665nm filter which results in an image of the Texas Red X labeled Internal Lane Standard 600 BIO Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 21 www promega com TMD016 Revised 7 15 7 Troubleshooting For questions not addressed here please contact
4. 17 6 E Controls 17 6 F Allelic Ladders 18 6 G Results 18 7 Troubleshooting 21 8 References 25 9 Appendix 27 9 A Advantages of Using the Loci in the PowerPlex 16 BIO System 27 9 B Power of Discrimination 31 9 C DNA Extraction and Quantitation Methods and Automation Support 32 9 D The Internal Lane Standard 600 BIO 32 9 E Preparing the PowerPlex 16 BIO System PCR Amplification Mix
5. 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 9 www promega com TMD016 Revised 7 15 7486MA 94 0 C 100 90 0 C 100 60 0 C 29 60 0 C 29 70 0 C 23 70 0 C 23 3 tmp 10 cycles 3 tmp 22 cycles Figure 1 The ramp rates for the GeneAmp PCR System 9700 thermal cycler 5 Detection of Amplified Fragments Using the Hitachi FMBIO II Fluorescence Imaging System Materials to Be Supplied by the User Solution compositions are provided in Section 9 F polyacrylamide gel electrophoresis apparatus dry heating block water bath or thermal cycler power supply 4 000 volt squaretooth comb 35cm 60 wells cut in half for 30 wells gel 0 4mm thick Owl Scientific Cat S2S 60A Nalgene tissue culture filter 0 2 micron aerosol resistant pipette tips Section 9 G low fluorescence glass plates 43cm x 19cm x 0 4mm The Gel Company Cat GG047 B0505S spacers 0 4mm clear spacers The Gel Company Cat SGR47 036 SA 43 Extension Lab Repco Cat 31096423 for use with 43cm glass plates clamps e g large office binder clamps 50 Long Ranger gel solution Cambrex Cat 50611 Long Ranger Singel pack for ABI sequencers 377 36cm Cambrex Cat 50691 or PAGE PLUS concentrate 40 solution Amresco Inc Cat E562 TBE 10X buffer 10 Ammonium Persulfate Cat V3131
6. 376 449 2 2 3 2 5 7 17 CSF1PO JOE 321 357 6 15 D16S539 JOE 264 304 5 8 15 D7S820 JOE 215 247 6 14 D13S317 JOE 176 208 7 15 D5S818 JOE 119 155 7 16 1Lengths of each allele in the allelic ladders have been confirmed by sequence analyses 2When using an internal lane standard such as the Internal Lane Standard 600 BIO calculated sizes of allelic ladder components may differ from those listed This occurs because different sequences in allelic ladder and ILS components may cause differences in migration The dye label also affects migration of alleles 3The alleles listed are those with a frequency of gt 1 1000 4For a current list of microvariants see the Variant Allele Report published at the U S National Institute of Standards and Technology NIST web site at www cstl nist gov div831 strbase 5Amelogenin is not an STR but displays a 106 base X specific band and a 112 base Y specific band 30 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD016 Revised 7 15 www promega com Table 8 The PowerPlex 16 BIO System Allele Determinations in Commonly Available Standard DNA Templates Standard DNA Templates1 STR Locus K5622 9947A 99483 2800M Penta E 5 14 12 13 11 11 7 14 D18S51 15 16 15 19 15 18 16 18 D21S11 29 30 31 30 30 29 30 29 31 2 TH01 9
7. 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 27 Sprecher C J et al 1996 A general approach to analysis of polymorphic short tandem repeat loci BioTechniques 20 266 76 28 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 29 Jones D A 1972 Blood samples Probability of discrimination J Forensic Sci Soc 12 355 9 30 Brenner C and Morris J W 1990 In Proceedings from the International Symposium on Human Identification 1989 Promega Corporation 21 53 31 Mandrekar P V Krenke B E and Tereba A 2001 DNA IQ The intelligent way to purify DNA Profiles in DNA 4 3 16 32 Krenke B E et al 2005 Development of a novel fluorescent two primer approach to quantitative PCR Profiles in DNA 8 1 3 5 Additional STR references can be found at www promega com geneticidentity Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 27 www promega com TMD016 Revised 7 15 9 Appendix 9 A Advantages of Using the Loci in the PowerPlex 16 BIO System The loci included in the PowerPlex 1
8. PowerPlex 16 Monoplex System Penta D JOE 100 reactions DC6651 Not For Medical Diagnostic Use Accessory Components Product Size Cat 2800M Control DNA 25 l DD7101 500 l DD7251 Gold STHR 10X Buffer 1 2ml DM2411 Nuclease Free Water 50ml P1193 Not For Medical Diagnostic Use Sample Preparation Systems Product Size Cat DNA IQ System 100 reactions DC6701 400 reactions DC6700 Differex System 50 samples DC6801 200 samples DC6800 Maxwell 16 Forensic Instrument 1 each AS3060 DNA IQ Reference Sample Kit for Maxwell 16 48 preps AS1040 DNA IQ Casework Pro Kit for Maxwell 16 48 preps AS1240 Plexor HY System 200 reactions DC1001 800 reactions DC1000 Slicprep 96 Device 10 pack V1391 Not for Medical Diagnostic Use Polyacrylamide Gel Electrophoresis Reagents Product Size Cat Ammonium Persulfate Molecular Grade 25g V3131 TBE Buffer 10X Molecular Biology Grade 1L V4251 Urea 1kg V3171 36 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD016 Revised 7 15 www promega com a PowerPlex 16 BIO System incorporates dye conjugates made with the Rhodamine Red X and Texas Red X fluorescent reactive dyes which are licensed from Molecular Probes Inc under U S Pat Nos 5 798 276 and 5 846 737 for DNA analysis Rhodamine Red i
9. The plates must be thoroughly cleaned before scanning Thoroughly clean both sides of the gel glass plate unit with deionized water and lint free paper before scanning Ethanol should not be used to clean the plate unit ethanol fluoresces and may be detected as background by the FMBIO instrument 3 The protocols in the data analysis section use the following filter setup 13137MA 1CH 598nm 3CH 505nm 2CH 665nm 4CH 577nm Filter Holder 1 Upper Filter Filter Holder 2 Lower Filter 14 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD016 Revised 7 15 www promega com 5 E Detection continued 4 Scan the gel using the parameters listed in Table 5 Use the 598nm filter Channel 1 to detect Rhodamine Red X labeled loci the 665nm filter Channel 2 to detect the Texas Red X labeled ILS 600 BIO the 505nm filter Channel 3 to detect the fluorescein labeled fragments and the 577nm filter Channel 4 to detect JOE labeled fragments Notes 1 Dust particles will be detected in the scan using the 577nm filter 2 The 598nm filter in Channel 1 is acceptable for autofocusing Table 5 Instrument Parameters for the Hitachi FMBIO II Fluorescence Imaging System and PowerPlex 16 BIO System Parameter Specification Material Type acrylamide gel Resolution Horizontal Vertical 150dpi 150dpi Rate N
10. Urea Cat V3171 TEMED bind silane methacryloxypropyltrimethoxysilane for use with squaretooth combs Liqui Nox detergent filter set for the PowerPlex 16 BIO System MiraiBio Cat 11999 246 00 5 A Polyacrylamide Gel Preparation Acrylamide Long Ranger gel solution 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 solutions 10 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD016 Revised 7 15 www promega com 5 A Polyacrylamide Gel Preparation continued 1 Thoroughly clean glass plates twice with hot water and a 1 Liqui Nox solution Rinse extremely well with deionized water Allow the glass plates to air dry in a dust free environment Dust particles will be detected in the scan using the 577 filter Use lint free paper e g Kimwipes tissues to clean the glass plates both before pouring the gel and prior to scanning Air from a bulb may also be used to remove dust If using a squaretooth comb one of the glass plates requires bind silane treatment The plates do not require special silane treatment when using a sharkstooth comb Bind Silane Treatment of Glass Plate Prepare fresh binding solution in a
11. shadow bands is due to the loss of a repeat unit during DNA amplification somatic variation within the DNA or both The amount of this artifact observed depends primarily on the locus and DNA sequence being amplified Terminal nucleotide addition 13 14 occurs when Taq DNA polymerase adds a nucleotide generally adenine to the 3 ends of amplified DNA fragments in a template independent manner 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 We have modified primer sequences and added a final extension step of 60 C for 30 minutes 15 to the amplification protocol to provide conditions for essentially complete terminal nucleotide addition when recommended amounts of DNA template are used The presence of microvariant alleles alleles differing from one another by lengths other than the repeat length complicates interpretation and assignment of alleles There appears to be a correlation between a high degree of polymorphism a tendency for microvariants and increased mutation rate 16 17 Thus FGA and D21S11 display numerous relatively common microvariants For reasons yet unknown the highly polymorphic Penta E locus does not display frequent microvariants Table 7 28 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 6
12. 2 a From the multicolor image zoom in on an area of medium intensity band to 200X or 300X b From the multicolor image find a medium intensity band in the channel color of interest c Switch to the black and white image for that channel d From the Gray Level Adjustment window highlight the Low Background percent box e Draw a box in the area directly above and almost on top of the medium intensity band Figure 2 Select Set This often results in a background above 90 Note Choose background while at 200 300X for each color f Do not adjust the signal Signal adjustment should be performed only if the signal is unusually high If signal needs adjustment highlight the High Signal percent box then select a band of medium intensity for signal adjustment Select Set 6 Repeat Step 5 for each of the other three colors by starting at the colored image and choosing the appropriate color band 7 Review the gel image in the Over Display Mode under Multi Lanes containing the Matrix 16 BIO sample should have this pattern repeated three times from the largest to smallest band red green blue and yellow Note Make sure that the channels are set as shown in Step 4 8 Perform color separation using the method described in Section 6 B or 6 C Enlarged view 3484TA07_1A Figure 2 Area of scan to choose for background adjustment 16 Promega Corporation 2800 Woods Hollow Road Mad
13. 22 with The Bode Technology Group Springfield VA North Carolina Bureau of Investigation Raleigh NC Palm Beach County Sheriff s Office West Palm Beach FL Virginia Division of Forensic Science Richmond VA and Charlotte Mecklenburg Police Department Laboratory NC Generation of these data includes analysis of over 200 individuals from African American Caucasian American and Hispanic American populations Data for Asian Americans includes analysis of over 150 individuals For additional population data for STR loci see references 23 28 and the Short Tandem Repeat DNA Internet DataBase at www cstl nist gov div831 strbase Table 9 shows the matching probability 29 for the PowerPlex 16 BIO System in various populations The matching probability of this system ranges from 1 in 1 83 1017 for Caucasian Americans to 1 in 1 41 1018 for 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 alleged father The typical paternity indices for the PowerPlex 16 BIO System are shown in Table 9 This system provides typical paternity indices exceeding 500 000 in each population group An alternative calculation used in paternity analyses is the power of exclusion 30 This value calculated for the system exceeds 0 999998 in all populations tested Table 9 Table 9 Matching P
14. 356 9526 608 274 4330 Fax 608 277 2516 23 www promega com TMD016 Revised 7 15 Symptoms Causes and Comments Bleedthrough Incorrect gray level Use matrix lanes for color separation Confirm that the bands selected are from the appropriate scanning channel Incorrect colors were assigned to the channels Repeat gray level adjustment Figure 2 Signal was too strong Use less template in the amplification reactions or load less amplified sample onto the gel Gel was not run long enough Bands become more diffuse less intense during the gel run Gels should be run until the 100 base fragment is near the bottom of the gel Imbalance of band intensities across loci Too much template The system is balanced using 1ng of DNA template Using greater than 2ng will lead to overrepresented smaller alleles and underrepresented larger alleles Use the recommended amount of template Alternatively reduce the number of cycles in the amplification program to improve locus to locus balance Poor quality polyacrylamide gel Prepare acrylamide and buffer solutions using high quality reagents We recommend 5 Long Ranger or 6 PAGE PLUS gels Too many cycles in the amplification protocol Use the recom mended amplification program confirm the number of cycles Too much enzyme present Use the recommended amount of AmpliTaq Gold DNA polymerase Degraded DNA sample Confirm the DNA integrity by running an aliquot on an agaros
15. Control DNA at 30 C to 10 C in a nonfrost free freezer Store the 2800M Control DNA at 2 to 10 C The PowerPlex 16 BIO 10X Primer Pair Mix PowerPlex 16 BIO Allelic Ladder Mix Matrix 16 BIO and Internal Lane Standard 600 BIO are light sensitive and must be stored in the dark We strongly recommend that pre amplification and post amplification reagents be stored and used separately with different pipettes tube racks etc 4 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD016 Revised 7 15 www promega com 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 9 10 The quality of the purified DNA as well as small changes in buffers ionic strength primer concentrations choice of thermal cycler and thermal cycling conditions can affect PCR success We suggest strict adherence to recommended procedures for amplification as well as for electrophoresis and fluorescence detection Additional research and validation are required if any modifications are made to the recommended protocols PCR based STR analysis is subject to contamination by minute amounts of human DNA Extreme care should be taken to avoid cross contamination when preparing sample DNA handling primer pairs assembling amplification reactio
16. below the bottom of the matrix bands 4 Select the single lane tool from the side menu and drag it down the matrix sample lane 5 Select Autoband on the side menu Make sure that 12 bands are autobanded 6 Select Color Separation from the Multi menu then select Multi A Multi Band Color Separator window will be displayed 7 Select Copy or highlight a stored pattern The pattern should have three sets of four colors with the pattern red green blue and yellow 8 If the pattern is not displayed correctly highlight the colored box under Use and select the correct color Note The Matrix 16 BIO pattern can be stored by highlighting Save as and typing in Matrix 16 BIO 9 Select OK 10 Select OK from the Multi Band Color Separator window Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 17 www promega com TMD016 Revised 7 15 6 D Autobanding 1 View and analyze the gel image to determine allele designations as recommended in the FMBIO user s manual For information regarding the use of FMBIO Analysis Software contact the Hitachi Technical Support Group 510 337 2000 or support miraibio com 2 We recommend the following autobanding parameters Parameter Default Setting Recommended Settings Starting Slope 2 5 1 5 2 0 Ending Slope 2 5 1 5 2
17. com TMD016 Revised 7 15 9 E Preparing the PowerPlex 16 BIO System PCR Amplification Mix Use Table 10 to calculate the required amount of each component of the PCR amplification mix Multiply the volume l per sample by the total number of reactions to obtain the final PCR amplification mix volume l Table 10 PCR Amplification Mix for the PowerPlex 16 BIO System PCR Master Mix Component Volume Per Sample Number of Reactions Final Volume l Gold STHR 10X Buffer 2 5 l PowerPlex 16 BIO 10X Primer Pair Mix 2 5 l AmpliTaq Gold DNA polymerase1 0 8 l 4u nuclease free water2 l Per tube template DNA volume2 0 5 1ng up to 19 2 l total reaction volume 25 l 1Assumes the AmpliTaq Gold DNA polymerase is at 5u l If the enzyme concentration is different the volume of enzyme used must be adjusted accordingly 2The PCR amplification mix volume added to the template DNA volume should total 25 l Consider the volume of template DNA and add nuclease free water to the PCR amplification mix to bring the final volume of the final reaction to 25 l 34 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD016 Revised 7 15 www promega com 9 F Composition of Buffers and Solutions 10 ammonium persulfate Add 0 05g of ammonium persulfate
18. is used This is most commonly observed with the vWA and D3S1358 amplification products Reduce the amount of template DNA used or reduce the number of cycles by 2 or 4 cycles 10 20 or 10 18 cycles Incorrect matrix pattern Incorrect filter set was used or filters in the wrong position for scanning Filters for scanning should be Position 1 598 Position 2 665 Position 3 505 Position 4 577 Incorrect colors were assigned to the channels Channel colors should be Channel 1 red Channel 2 blue Channel 3 green Channel 4 yellow Extra bands visible in one or all color channels Contamination with another template DNA or previously amplified DNA Cross contamination can be a problem Use aerosol resistant pipette tips and change gloves regularly Artifacts of STR amplification Amplification of STRs sometimes generates artifacts that appear as faint peaks one repeat unit smaller than the allele Stutter band peak heights will be high if the samples are overloaded Allelic ladder contamination Store pre and post amplification components separately Samples were not completely denatured Heat denature samples at 95 C for 2 minutes and immediately chill on crushed ice or in an ice water bath prior to loading the gel Do not cool the samples in a thermal cycler set at 4 C as this may lead to artifacts due to DNA re annealing Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800
19. with water 10 Allow polymerization to proceed for at least 1 hour Check the polymerization control to be sure that polymer ization has occurred Note The gel may be stored overnight if a paper towel saturated with deionized water and plastic wrap are placed around the top and bottom to prevent the gel from drying out crystallization of the urea will destroy the gel 5 B Gel Pre Run 1 Remove the clamps from the polymerized acrylamide gel If necessary clean any excess acrylamide from the glass plates with Kimwipes tissues saturated with deionized water 2 Shave any excess polyacrylamide away from the comb remove the comb 3 Add 1X TBE buffer to the bottom chamber of the electrophoresis apparatus 4 Gently lower the gel glass plates unit into the buffer with the longer plate facing out and the well side on top 5 Secure the glass plates to the gel electrophoresis apparatus 6 Add 1X TBE buffer to the top chamber of the electrophoresis apparatus 7 Using a 50 100cc syringe filled with buffer remove any air bubbles on the top of the gel Be certain the well area is devoid of air bubbles and small pieces of polyacrylamide Using a syringe with a bent 18 gauge needle remove any air bubbles from 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 referen
20. your local Promega Branch Office or Distributor Contact information available at www promega com E mail genetic promega com Symptoms Causes and Comments Faint or absent allele bands Impure template DNA Because of the small amount of template used this is rarely a problem Depending on the DNA extraction procedure used and sample source inhibitors may be present in the DNA sample Diluting the template in TE 4 buffer or water prior to amplification may improve results Insufficient template DNA Use the recommended amount of template DNA Insufficient enzyme activity Use the recommended amount of AmpliTaq Gold DNA polymerase Check the expiration date on the tube label Incorrect amplification program Confirm the amplification program 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 also may affect PCR Store DNA in TE 4 buffer 10mM Tris HCl pH 8 0 0 1mM EDTA or nuclease free water Primer concentration was too low Use the recommended primer concentration Vortex the PowerPlex 16 BIO 10X Primer Pair Mix for 15 seconds before use Thermal cycler plate or tube problems Review the thermal cycling protocols in Section 4 B We have not tested other reaction tub
21. 0 Duration 0 2 0 1 Noise Level 20 15 30 3 The data from the four color analysis should contain the following Channel Filter Display as Color Dye Label Loci 1 598nm Red Rhodamine Red X FGA TPOX D8S1179 vWA Amelogenin 2 665nm Blue Texas Red X Internal Lane Standard 600 BIO 3 505nm Green Fluorescein Penta E D18S51 D21S11 TH01 D3S1358 4 577nm Yellow JOE Penta D CSF1PO D16S539 D7S820 D13S317 D5S818 6 E Controls Observe the lanes containing the negative controls Using the protocols defined in this manual the negative controls should be devoid of amplification products Observe the lanes containing the 2800M Control DNA Compare the 2800M Control DNA allelic repeat sizes with the locus specific allelic ladder The expected 2800M Control DNA allele designations for each locus are listed in Table 8 Section 9 A 18 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD016 Revised 7 15 www promega com 6 F Allelic Ladders In general allelic ladders contain fragments of the same lengths as either most or all known alleles for the locus Allelic ladder sizes and repeat units are listed in Table 7 Section 9 A Visual comparison between allelic ladder and amplified samples of the same locus allows precise assignment of alleles Analysis using specific instrumentation also allows allele determi
22. 08 277 2516 TMD016 Revised 7 15 www promega com Table 6 The PowerPlex 16 BIO System Locus Specific Information STR Locus Label Chromosomal Location GenBank Locus and Locus Definition Repeat Sequence1 5 3 Penta E FL 15q NA AAAGA D18S51 FL 18q21 3 HUMUT574 AGAA 18 D21S11 FL 21q11 21q21 HUMD21LOC TCTA Complex 18 TH01 FL 11p15 5 HUMTH01 Human tyrosine hydroxylase gene AATG 18 D3S1358 FL 3p NA TCTA Complex FGA Rhodamine Red X 4q28 HUMFIBRA Human fibrinogen alpha chain gene TTTC Complex 18 TPOX Rhodamine Red X 2p24 2pter HUMTPOX Human thyroid peroxidase gene AATG D8S1179 Rhodamine Red X 8q NA TCTA Complex 18 vWA Rhodamine Red X 12p12 pter HUMVWFA31 Human von Willebrand factor gene TCTA Complex 18 Amelogenin2 Rhodamine Red X Xp22 1 22 3 and Y HUMAMEL Human Y chromosomal gene for Amelogenin like protein NA Penta D JOE 21q NA AAAGA CSF1PO JOE 5q33 3 34 HUMCSF1PO Human c fms proto oncogene for CSF 1 receptor gene AGAT D16S539 JOE 16q24 qter NA GATA D7S820 JOE 7q11 21 22 NA GATA D13S317 JOE 13q22 q31 NA TATC D5S818 JOE 5q23 3 32 NA AGAT 1The August 1997 report 19 20 of the DNA Commission of the International Society for Forensic Haemogenetics ISFH states 1 for STR loci within coding genes the coding strand shall be used and the repeat sequence motif defined using the fi
23. 11 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 13 www promega com TMD016 Revised 7 15 5 D Gel Electrophoresis 1 After loading run the gel using the same conditions as in Section 5 B Observe the lane containing Gel Tracking Dye to monitor sample migration In a 5 Long Ranger acrylamide gel xylene cyanol dye migrates at approxi mately 190 bases and in a 6 PAGE PLUS acrylamide gel xylene cyanol migrates at approximately 130 bases In both gels bromophenol blue migrates at less than 80 bases 2 Based on size ranges for each locus Table 7 and migration characteristics of the dyes contained in the Gel Tracking Dye stop electrophoresis before the smallest locus i e Amelogenin has reached the bottom of the gel Note Some rare alleles i e 9 repeats for the D3S1358 locus are close to 100 bases in size The gel should be stopped before the 100 base fragment of the ILS 600 BIO has reached the bottom Table 4 Recommended Run Times for SA 43 Gels Gel Type Leading Edge of Xylene Cyanol distance from bottom of gel Approximate Run Time 60 Watts 5 Long Ranger 17 5cm 1 hour 30 minutes 6 PAGE PLUS 9 5cm 2 hours These recommendations are based on a one hour pre run Run time and xylene cyanol migration will vary with length of pre run 5 E Detection 1 After electrophoresis remove the gel glass plate unit from the apparatus Do not separate the glass plates 2
24. 3 9 3 8 9 3 6 9 3 6 9 3 D3S1358 16 16 14 15 15 17 17 18 FGA 21 24 23 24 24 26 20 23 TPOX 8 9 8 8 8 9 11 11 D8S1179 12 12 13 13 12 13 14 15 vWA 16 16 17 18 17 17 16 19 Amelogenin X X X X X Y X Y Penta D 9 13 12 12 8 12 12 13 CSF1PO 9 10 10 12 10 11 12 12 12 D16S539 11 12 11 12 11 11 9 13 D7S820 9 11 10 11 11 11 8 11 D13S317 8 8 11 11 11 11 9 11 D5S818 11 12 11 11 11 13 12 12 1Information on strains K562 9947A and 9948 is available online at http ccr coriell org Strain K562 is available from the American Type Culture Collection www atcc org Manassas VA Information about the use of 9947A and 9948 DNA as standard DNA templates can be found in reference 21 2Strain K562 displays three alleles at the D21S11 locus 3Strain 9948 displays three alleles at the CSF1PO locus The peak height for allele 12 is much lower than those for alleles 10 and 11 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 31 www promega com TMD016 Revised 7 15 9 B Power of Discrimination The fifteen STR loci amplified with the PowerPlex 16 BIO System provide powerful discrimination Population statistics for these loci and their various multiplex combinations are displayed in Table 9 These data were developed as part of a collaboration
25. 6 total volume 50ml 1Long Ranger Singel Packs may also be used Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 11 www promega com TMD016 Revised 7 15 4 Filter the acrylamide solution through a 0 2 micron filter and pour into a squeeze bottle 5 Add the appropriate amounts of TEMED and 10 ammonium persulfate to the acrylamide solution and mix gently Component 5 Long Ranger Gel 50ml TEMED 18 0g 10 ammonium persulfate 26 0ml 6 Pour the gel by starting at the well end of the plates and carefully pouring the acrylamide between the horizontal glass plates Allow the solution to fill the top width of the plates Slightly tilt the plates to assist movement of the solution to the bottom of the plates while maintaining a constant flow of solution When the solution begins to flow out from the bottom position the plates horizontally 7 Insert the squaretooth comb between the glass plates until the teeth are almost completely inserted into the gel or insert one 14cm doublefine 49 point sharkstooth comb straight side into the gel between the glass plates 6mm of the comb should be between the two glass plates 8 Secure the comb with three evenly spaced clamps 9 Pour the remaining acrylamide solution into a disposable conical tube as a polymerization control Rinse the squeeze bottle including the spout
26. 6 BIO System Tables 6 7 and 8 were selected because they satisfy the needs of several major standardization bodies throughout the world For example the United States Federal Bureau of Investigation FBI has selected 13 STR core loci for typing prior to searching or including submitting samples in the Combined DNA Index System CODIS the U S national database of convicted offender profiles The PowerPlex 16 BIO System amplifies all CODIS core loci in a single reaction The PowerPlex 16 BIO System also contains two low stutter highly polymorphic pentanucleotide repeat loci Penta E and Penta D These additional loci add significantly to the discrimination power of the system making the PowerPlex 16 BIO System a single amplification system with a power of exclusion sufficient to resolve paternity disputes definitively In addition the extremely low stutter seen with Penta E and Penta D makes them ideal loci to evaluate DNA mixtures often encountered in forensic casework Finally the Amelogenin locus is included in the PowerPlex 16 BIO System to allow gender identification of each sample Table 8 lists the system alleles revealed in commonly available standard DNA templates We have carefully selected STR loci and primers to avoid or minimize artifacts including those associated with Taq DNA polymerase such as repeat slippage and terminal nucleotide addition Repeat slippage 11 12 sometimes called n 4 bands stutter or
27. A Repeat 256 times Gray Level Correction Type range Cutoff Threshold Low background High signal 50 1 Reading Sensitivity 100 598nm channel 100 665nm channel 100 505nm channel 100 577nm channel Focusing Point1 0 0mm NA not applicable 1Focusing point of 0 0mm is based on use of 5mm glass plates If using precast gels or thinner glass plates the focusing point may need to be adjusted Optimal focusing point may vary from instrument to instrument Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 15 www promega com TMD016 Revised 7 15 6 Data Analysis Four color separation requires FMBIO Analysis Software Macintosh Version 8 0 or higher The procedures included in this manual are suggestions for color separation Further optimization may be needed for individual laboratories 6 A Background Adjustment 1 Create a new project using the raw data files 2 Go to Multi and switch the display mode to over 3 Enlarge the project window to display four channels Note The initial multicolor image may not be displayed as four colors 4 Highlight and change the colors in the boxes so that Channel 1 is red Channel 2 is blue Channel 3 is green and Channel 4 is yellow 5 Perform background adjustment prior to multicolor separation Adjust the background for each channel Figure
28. Revised 7 15 TMD016 T E C H N I C A L M A N U A L PowerPlex 16 BIO System Instructions for Use of Product DC6540 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 1 www promega com TMD016 Revised 7 15 All technical literature is available at www promega com protocols Visit the web site to verify that you are using the most current version of this Technical Manual E mail Promega Technical Services if you have questions on use of this system techserv promega com PowerPlex 16 BIO System 1 Description 2 2 Product Components and Storage Conditions 3 3 Before You Begin 4 4 Protocols for DNA Amplification Using the PowerPlex 16 BIO System 5 4 A Amplification Setup 5 4 B Amplification Thermal Cycling 7 5 Detection of Am
29. System contains the loci Penta E D18S51 D21S11 TH01 D3S1358 FGA TPOX D8S1179 vWA Amelogenin Penta D CSF1PO D16S539 D7S820 D13S317 and D5S818 In the system one primer specific for Penta E D18S51 D21S11 TH01 and D3S1358 is labeled with fluorescein FL one primer specific for FGA TPOX D8S1179 vWA and Amelogenin is labeled with Rhodamine Red X and one primer specific for Penta D CSF1PO D16S539 D7S820 D13S317 and D5S818 is labeled with 6 carboxy 4 5 dichloro 2 7 dimethoxy fluorescein JOE All sixteen loci are amplified simultaneously in a single tube and analyzed in a single gel lane The PowerPlex 16 Monoplex System Penta E Fluorescein Cat DC6591 and PowerPlex 16 Monoplex System Penta D JOE Cat DC6651 are available to amplify the Penta E and Penta D loci respectively Each monoplex system allows amplification of a single locus to confirm results obtained with the PowerPlex 16 System PowerPlex 16 BIO System or PowerPlex 2 1 System The monoplex systems also can be used to re amplify DNA samples when one or more of the loci do not amplify initially due to nonoptimal amplification conditions or poor DNA quality The PowerPlex 16 BIO System is designed specifically for use with the Hitachi FMBIO II Fluorescence Imaging System It provides all of the materials necessary to amplify STR regions of purified human genomic DNA except for AmpliTaq Gold DNA polymerase This manual contains separate p
30. agment off the bottom of the gel If necessary run the gel for additional time after the first scan then scan the gel a second time to achieve greater separation of larger alleles Scanning resolution was too low Default scanning resolution is 150dpi If necessary this resolution can be increased to 300dpi which should help sharpen bands White background with low signal intensity Part of the white spacers were scanned Rescan the gel being careful not to scan any portion of the spacers remove the spacers for scanning or use black electrical tape to cover the spacers We recommend using clear spacers Dark grainy background with low signal intensity Focusing point may need to be adjusted Perform multiple scans using different focusing points Adjust focusing point if necessary Plates were improperly washed Improper washing of plates can cause a soap residue to build up on the plates which can cause background fluorescence Plate may be soaked in 1N NaOH to remove residue Do not use ethanol to clean plates prior to scanning Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 25 www promega com TMD016 Revised 7 15 8 References 1 Edwards A et al 1991 DNA typing with trimeric and tetrameric tandem repeats Polymorphic loci detection systems and population genetics In The Second International Symposium on Human Identifica
31. al 1991 Guidelines for a quality assurance program for DNA analysis Crime Laboratory Digest 18 44 75 11 Levinson G and Gutman G A 1987 Slipped strand mispairing A major mechanism for DNA sequence evolution Mol Biol Evol 4 203 21 12 Schlotterer C and Tautz D 1992 Slippage synthesis of simple sequence DNA Nucl Acids Res 20 211 5 13 Smith J R et al 1995 Approach to genotyping errors caused by nontemplated nucleotide addition by Taq DNA polymerase Genome Res 5 312 7 14 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 15 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 16 Moller A Meyer E and Brinkmann B 1994 Different types of structural variation in STRs HumFES FPS HumVWA and HumD21S11 Int J Leg Med 106 319 23 26 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD016 Revised 7 15 www promega com 8 References continued 17 Brinkmann B Moller A and Wiegand P 1995 Structure of new mutations in 2 STR systems Int J Leg Med 107 201 3 18 Griffiths R et al 1998 New reference allel
32. als The DNA IQ System has been tested with the PowerPlex Systems to ensure a streamlined process See Section 9 G for ordering information For applications requiring human specific DNA quantification the Plexor HY System Cat DC1001 DC1000 has been developed 32 See Section 9 G for ordering information For information about automation of Promega chemistries on automated workstations using Identity Automation solutions contact your local Promega Branch Office or Distributor contact information available at www promega com support worldwide contacts e mail genetic promega com or visit www promega com idautomation 9 D The Internal Lane Standard 600 BIO The Internal Lane Standard ILS 600 BIO contains 21 DNA fragments of 80 100 120 140 160 180 200 225 250 275 300 325 350 375 400 425 450 475 500 550 and 600 bases in length Each fragment is labeled with Texas Red X and may be detected separately as a fourth color in the presence of PowerPlex 16 BIO amplified material using the Hitachi FMBIO II Fluorescence Imaging System The ILS 600 BIO is designed for use in each gel lane to increase precision in analyses when using the PowerPlex 16 BIO System A protocol for preparation and use of this internal lane standard is provided in Section 5 C Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 33 www promega
33. an Fluorescein Penta E D18S51 D21S11 TH01 D3S1358 B L 1 2 3 4 5 L 577nm Scan JOE Penta D D16S539 CSF1PO D7S820 D13S317 D5S818 C L 1 2 3 4 5 L 598nm Scan Rhodamine Red X FGA D8S1179 TPOX vWA Amel Figure 3 The PowerPlex 16 BIO System Five single source DNA samples lanes 1 5 were amplified with the PowerPlex 16 BIO Primer Pair Mix using 1ng of DNA template Lane 1 shows the results using 1ng of 9947A DNA Lanes labeled L contain allelic ladders for each of the sixteen loci contained in the system All materials were separated using a 6 PAGE PLUS acrylamide gel and detected using the Hitachi FMBIO II Fluorescence Imaging System Panel A A scan using a 505nm filter showing the fluorescein labeled loci Penta E D18S51 D21S11 TH01 and D3S1358 Panel B A scan using a 577nm filter showing the JOE labeled loci Penta D CSF1PO D16S539 D7S820 D13S317 and D5S818 Panel C A scan using a 598nm filter showing the Rhodamine Red X labeled loci FGA TPOX D8S1179 vWA and Amelogenin 20 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD016 Revised 7 15 www promega com 6331TA D 600 550 500 475 450 425 400 375 350 325 300 275 250 225 200 180 160 140 120 100 665nm Scan Texas Red X C L 1 2 3 4 5 598nm Scan
34. ation mix for all samples It also ensures that each reaction contains the same PCR amplification mix 3 Place one clean 0 2ml or 0 5ml reaction tube for each reaction into a rack and label appropriately Alternatively use a MicroAmp plate and label appropriately 4 Add the final volume of each reagent listed in Table 2 into a sterile tube Mix gently Table 2 shows the component volumes per reaction A worksheet to calculate the required amount of each PCR amplification mix component is provided in Section 9 E Table 10 Table 2 PCR Amplification Mix for the PowerPlex 16 BIO System PCR Amplification Mix Component1 Volume Per Sample nuclease free water to a final volume of 25 0 l Gold STHR 10X Buffer 2 5 l PowerPlex 16 BIO 10X Primer Pair Mix 2 5 l AmpliTaq Gold DNA polymerase2 0 8 l 4u template DNA 0 5 1ng 3 up to 19 2 l total reaction volume 25 l 1Add nuclease free water to the PCR amplification mix first then add Gold STHR 10X Buffer PowerPlex 16 BIO 10X Primer Pair Mix and AmpliTaq Gold DNA polymerase The template DNA will be added at Step 6 2Assumes AmpliTaq Gold DNA polymerase is at 5u l If the concentration is different the volume of enzyme used must be adjusted accordingly 3Store DNA templates in nuclease free water or TE 4 buffer 10mM Tris HCl pH 8 0 0 1mM EDTA If the DNA template is stored in TE buffer that is not pH 8 0 or contains a higher EDTA concentrati
35. ce we generally use 60 watts for 45 60 minutes for a 43cm gel The gel running conditions may need to be adjusted to reach a temperature of 50 C 9 Prepare samples and allelic ladder samples during the gel pre run 12 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD016 Revised 7 15 www promega com 5 C Sample Preparation and Loading The Internal Lane Standard 600 ILS BIO is included in the PowerPlex 16 BIO System for use as an internal size marker With this approach only 2 3 lanes of the PowerPlex 16 BIO Allelic Ladder Mix are required per gel The Matrix 16 BIO is included with the system to aid in color separation The Matrix 16 BIO should be run in one or two lanes per gel 1 Prepare a loading cocktail by combining and mixing the ILS 600 BIO and Bromophenol Blue Loading Solution as follows Volume Per Sample Number of Lanes Total Volume Internal Lane Standard BIO 1 l Bromophenol Blue Loading Solution 3 l 2 Vortex for 10 15 seconds 3 Combine 4 l of prepared loading cocktail and 2 l of amplified sample or PowerPlex 16 BIO Allelic Ladder Mix Vortex the allelic ladder prior to pipetting 4 For matrix lanes combine 2 5 l of Matrix 16 BIO and 2 5 l of Bromophenol Blue Loading Solution Note If the fluorescent signal is too intense dilute samples in Gold STHR 1X Buffer bef
36. chemical fume hood by adding 3 l of bind silane to a 1 5ml microcentrifuge tube containing 1 0ml of 0 5 acetic acid in 95 ethanol Wipe the short plate with a Kimwipes tissue saturated with freshly prepared binding solution Wait 5 minutes for the binding solution to dry Wipe the comb area of the glass plate 5 6 times with 95 ethanol and Kimwipes tissues to remove excess binding solution 2 Assemble the glass plates by placing 0 4mm side spacers between the front and rear glass plates using binder clamps to hold them in place 3 4 clamps on each side A bottom spacer is neither required nor recommended Place the assembly horizontally on a test tube rack or similar support Note We recommend clear spacers If white or opaque spacers are used place black electrical tape on the longer plate over the spacer area to prevent the spacers from being included in the scan which can affect the signal and background 3 Prepare a 5 Long Ranger or 6 PAGE PLUS acrylamide solution by combining the ingredients listed in Table 3 Table 3 Preparation of 5 Long Ranger Polyacrylamide Gels Component 5 Gel Final Concentration urea 18 0g 6M deionized water 26 0ml 10X TBE Buffer 5 0ml 1X 50 Long Ranger gel solution1 5 0ml 5 total volume 50ml Component 6 PAGE PLUS Gel Final Concentration urea 18g 6M deionized water 24ml 10X TBE buffer 5ml 1X 40 PAGE PLUS gel solution 7 5ml
37. e gel Repurify the template DNA if necessary Miscellaneous balance problems Thaw the 10X Primer Pair Mix and Gold STHR 10X Buffer completely and vortex for 15 seconds before using Calibrate thermal cyclers and pipettes routinely Decreasing the annealing temperatures by 1 2 C also can improve the balance 24 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD016 Revised 7 15 www promega com 7 Troubleshooting continued Symptoms Causes and Comments Imbalance of band intensities across loci Too much template from card or membrane punches of continued bloodstains Cards or membranes that bind DNA tightly can contain more template DNA than recommended This will lead to overrepresented smaller alleles and underrepresented larger alleles Use the recommended amount of template by using a smaller punch of the membrane Alternatively fewer cycles of amplification can compensate for this type of unevenness of product yield i e 10 20 or 10 18 cycling Incorrect plates or tubes were used Use only MicroAmp tubes or plates Due to potential differences in heat transfer capabilities plates or tubes from other manufacturers might result in imbalance Poor separation of alleles in ladder lanes or Gel was not run long enough Run gel as long as possible difficulty resolving microvariant alleles without running the 100 base ILS fr
38. emark of Flinders Technologies Pty Ltd and is licensed to Whatman GenBank is a registered trademark of the U S Dept of Health and Human Services Kimwipes is a registered trademark of Kimberly Clark Corporation Liqui Nox is a registered trademark of Alconox Inc Long Ranger and Long Ranger Singel are registered trademarks of Cambrex Corporation Macintosh is a registered trademark of Apple Computer Inc MicroAmp is a registered trademark of Applera Corporation Nalgene is a registered trademark of Nalge Nunc International PAGE PLUS is a trademark of Amresco Inc Rhodamine Red is a trademark of Molecular Probes Inc Texas Red is a registered trademark of Molecular Probes Inc Triton is a registered trademark of Union Carbide Chemicals amp Plastics Technology Corporation 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 9 H Summary of Changes The following changes were made to the 7 15 revision of this document 1 The patent license statements were updated 2 The discontinued 100 reaction size of the product was removed 3 The document design was updated
39. ermal Cycler 95 C for 11 minutes then 96 C for 1 minute then 94 C for 30 seconds ramp 68 seconds to 60 C hold for 30 seconds ramp 50 seconds to 70 C hold for 45 seconds for 10 cycles then 90 C for 30 seconds ramp 60 seconds to 60 C hold for 30 seconds ramp 50 seconds to 70 C hold for 45 seconds for 22 cycles then 60 C for 30 minutes 4 C soak 95 C for 11 minutes then 96 C for 2 minutes then 94 C for 1 minute 60 C for 1 minute 70 C for 1 5 minutes for 10 cycles then 90 C for 1 minute 60 C for 1 minute 70 C for 1 5 minutes for 22 cycles then 60 C for 30 minutes 4 C soak 1When using the GeneAmp PCR System 9700 thermal cycler the ramp rates indicated in the cycling program must be set and the program must be run in 9600 ramp mode The ramp rates are set in the ramp rate modification screen While viewing the cycling program navigate to the ramp rate modification screen by selecting More then Modify On the ramp rate modification screen the default rates for each step are 100 The rate under each hold step is the rate at which the temperature will change to that hold temperature Figure 1 shows the ramp rates for the GeneAmp PCR System 9700 thermal cycler The ramp mode is set after start has been selected for the thermal cycling run A Select Method Options screen appears Select 9600 ramp mode and enter the reaction volume Promega Corporation
40. es plates or thermal cyclers Calibrate the thermal cycler heating block if necessary Samples were not denatured completely Heat denature samples at 95 C for 2 minutes and immediately chill on crushed ice or in an ice water bath prior to loading the gel Do not cool the samples in a thermal cycler set at 4 C as this may lead to artifacts due to DNA re annealing Background level was too high 99 in the black and white image in question Repeat the color separation process starting with the gray level adjustment and using raw data OR lower the background to a percentage equivalent to the percentage that was set in the other three images 22 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD016 Revised 7 15 www promega com 7 Troubleshooting continued Symptoms Causes and Comments Bands are fuzzy throughout the lanes Poor quality polyacrylamide gel Prepare acrylamide and buffer solutions using high quality reagents We recommend 5 Long Ranger or 6 PAGE PLUS gels Gel pre run was not long enough Pre run the gel until the gel temperature is 50 C Electrophoresis temperature was too high Run gel at lower temperature 40 50 C n 1 bands present Following amplification lengthen the final extension step from 30 minutes at 60 C to 45 minutes n 1 bands may be generated when more than 1ng of template DNA
41. ic ladders to improve allelic designation in a multiplex STR system Int J Legal Med 111 267 72 19 B r 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 Legal Med 110 175 6 20 Gill P et al 1997 Considerations from the European DNA profiling group EDNAP concerning STR nomen clature Forensic Sci Int 87 185 92 21 Fr geau C J et al 1995 Characterization of human lymphoid cell lines GM9947 and GM9948 as intra and interlaboratory reference standards for DNA typing Genomics 28 184 97 22 Levadokou E et al 2001 Allele frequencies for fourteen STR loci of the PowerPlex 1 1 and 2 1 Multiplex Systems and Penta D Locus in Caucasians African Americans Hispanics and other populations of the United States of America and Brazil J Forensic Sci 46 736 61 23 Lins A M et al 1998 Development and population study of an eight locus short tandem repeat STR multi plex system J Forensic Sci 43 1168 80 24 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 Human Genet 53 953 8 25 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 26
42. ison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD016 Revised 7 15 www promega com 6 B Color Separation Using Individual Bands Perform the multicolor separation using individual bands from the Matrix 16 BIO lane s The Matrix 16 BIO sample should have this pattern repeated three times from the largest to smallest band red green blue and yellow 1 From the multicolor image zoom in on matrix lane to 200X or 300X 2 Highlight Color Separation under Multi A color separation window will be displayed and the Basis Image shown will be red 3 Draw a box around a red band top band of the matrix avoiding as much background as possible and select Set 4 Repeat Steps 2 and 3 for each of the other colors by changing the Basis Image to the color of the band that is highlighted 5 Select OK to generate the separated images 6 Select OK when asked if you want to execute color separation 6 C Color Separation Using Bands In a Single Lane The multicolor separation can be done using a single lane Use the Matrix 16 BIO lane Read the section on image analysis tools in the FMBIO user s manual before creating a color separation matrix with the Multi Band Color Separator 1 Select 1 D Gel under Tools 2 Select Layer and highlight filename 2CH Select OK 3 Move the range to just above the top and just
43. n water 8 For the negative amplification control pipet nuclease free water or TE 4 buffer instead of template DNA into a reaction tube containing PCR amplification mix 9 If using the GeneAmp PCR System 9600 9700 or 2400 thermal cycler and MicroAmp reaction tubes or plates no addition of mineral oil to the reaction tubes is required However if using the model 480 thermal cycler and GeneAmp reaction tubes add one drop of mineral oil to each tube before closing 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 10 Optional Briefly centrifuge the tubes to bring contents to the bottom and remove any air bubbles 4 B Amplification Thermal Cycling This manual contains protocols for use of the PowerPlex 16 BIO System with the Perkin Elmer model 480 and GeneAmp PCR system 9600 9700 and 2400 thermal cyclers For information about other thermal cyclers please contact Promega Technical Services by e mail genetic promega com Amplification and detection instrumentation may vary Testing at Promega shows that 10 22 cycles work well for 0 5 1ng of purified DNA templates For higher amounts of input DNA or to decrease sensitivity fewer cycles such as 10 16 10 18 or 10 20 should be evaluated In house validation should be performed 1 Place the tubes or MicroAmp plate in the thermal cycler 2 Select and run a recommended
44. nation by comparing amplified sample fragments with either allelic ladders internal size standards or both see software documentation from instrument manufacturer When using an internal lane standard the calculated lengths of allelic ladder components will differ from those listed in Table 7 This is due to differences in migration resulting from sequence differences between allelic ladder fragments and internal lane standard fragments Dye labels also affect fragment migration Note It may prove helpful to confirm that your gel analysis software identifies the correct number of alleles present in the allelic ladder lanes prior to analysis of sample lanes The PowerPlex 16 BIO Allelic Ladder has 86 alleles in the fluorescein channel 20 Penta E alleles 22 D18S51 alleles 25 D21S11 alleles 10 TH01 alleles and 9 D3S1358 alleles 61 alleles in the JOE channel 14 Penta D alleles 10 CSF1PO alleles 9 D16S539 alleles 9 D7S820 alleles 9 D13S317 alleles and 10 D5S818 alleles and 55 alleles in the Rhodamine Red X channel 20 FGA alleles 8 TPOX alleles 12 D8S1179 alleles 13 vWA alleles and 2 Amelogenin alleles 6 G Results Representative results using the PowerPlex 16 BIO System are shown in Figures 3 and 4 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 19 www promega com TMD016 Revised 7 15 6330TA A L 1 2 3 4 5 L 505nm Sc
45. ns and analyzing amplification products Reagents and materials used prior to amplification Gold STHR 10X Buffer 2800M Control DNA and PowerPlex 16 BIO 10X Primer Pair Mix are provided in a separate box and should be stored separately from those used following amplification PowerPlex 16 BIO Allelic Ladder Mix Internal Lane Standard 600 BIO Matrix 16 BIO Bromophenol Blue Loading Solution and Gel Tracking Dye Always include a negative control reaction i e no template to detect reagent contamination We highly recommend the use of gloves and aerosol resistant pipette tips e g ART tips Section 9 G 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 Reagents Hazards acrylamide suspected carcinogen toxic ammonium persulfate oxidizer corrosive formamide contained in the Bromophenol Blue Loading Solution irritant teratogen TEMED corrosive flammable urea irritant Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 5 www promega com TMD016 Revised 7 15 4 Protocols for DNA Amplification Using the PowerPlex 16 BIO System Materials to Be Supplied by the User model 480 or GeneAmp system 9600 9700 or 2400 thermal cycler Applied Biosystems
46. on the volume of DNA added should not exceed 20 of the final reaction volume Amplification efficiency and quality can be greatly altered by changes in pH due to added Tris HCl available magnesium concentration due to chelation by EDTA or other PCR inhibitors which may be present at low concentrations depending on the source of the template DNA and the extraction procedure used Amplification of gt 2ng of DNA template results in an imbalance in band intensities from locus to locus The smaller loci show greater amplification yield than larger loci Reducing the number of cycles in the amplification program by 2 to 4 cycles i e 10 20 or 10 18 cycling can improve locus to locus balance Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 7 www promega com TMD016 Revised 7 15 5 Pipet PCR amplification mix into each reaction tube 6 Pipet each template DNA 0 5 1ng into the respective tube containing PCR amplification mix 7 For the positive amplification control vortex the tube of 2800M Control DNA then dilute an aliquot to 0 5ng or 1ng in the desired template DNA volume Pipet 0 5 1ng of the diluted DNA into a microcentrifuge tube containing PCR amplification mix Note To store diluted 2800M Control DNA dilute the DNA to 0 5ng l in TE 4 buffer with 20 g ml glycogen and store at 4 C Do not store dilutions performed i
47. on setup Use equipment and supplies dedicated for amplification setup Meticulous care must be taken to ensure successful amplification A guide to amplification troubleshooting is provided in Section 7 The concentration of 2800M Control DNA was determined by measuring absorbance at 260nm Quantification of this control DNA by other methods such as qPCR may result in a different value Prepare a fresh DNA dilution for each set of amplifications Do not store diluted DNA e g 0 25ng l or less 1 Thaw Gold STHR 10X Buffer and PowerPlex 16 BIO 10X Primer Pair Mix Notes 1 Mix reagents by vortexing for 15 seconds before each use Do not centrifuge the 10X Primer Pair Mix after vortexing as this may cause the primers to be concentrated at the bottom of the tube 2 A precipitate may form in Gold STHR 10X Buffer If this occurs warm the buffer briefly at 37 C then vortex until the precipitate is in solution 6 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD016 Revised 7 15 www promega com 4 A Amplification Setup continued 2 Determine the number of reactions to be set up This should include positive and negative control reactions Add 1 or 2 reactions to this number to compensate for pipetting error While this approach does consume a small amount of each reagent it ensures that you will have enough PCR amplific
48. ore mixing with loading cocktail or use less DNA template in the amplification reactions 5 Optional Place 2 l of Gel Tracking Dye and 3 l of Gold STHR 1X Buffer in one tube The Gel Tracking Dye contains both bromophenol blue and xylene cyanol This dye is loaded in the outermost lane of the gel at least 2 lanes from the nearest sample and is used as a visual indicator of migration Note The xylene cyanol dye in the Gel Tracking Dye will fluoresce when scanned with the Hitachi FMBIO II Fluorescence Imaging System Leave at least two empty lanes between the Gel Tracking Dye and sample lanes 6 Briefly centrifuge samples to bring contents to the bottom of the tubes 7 Just prior to loading the gel denature samples by heating at 95 C for 2 minutes and immediately chill on crushed ice or in an ice water bath 8 After the pre run use a 50 100cc syringe filled with buffer and fitted with a bent 18 gauge needle to flush urea from the well area If using a squaretooth comb do not reinsert the comb as the samples will be loaded directly into the wells If using a sharkstooth comb insert the teeth into the gel approximately 1 2mm and leave the comb inserted in the gel during both gel loading and electrophoresis 9 Load 3 l of each denatured sample into the respective wells The loading process should take no longer than 20 minutes to prevent the gel from cooling Promega Corporation 2800 Woods Hollow Road Madison WI 537
49. plified Fragments Using the Hitachi FMBIO II Fluorescence Imaging System 9 5 A Polyacrylamide Gel Preparation 9 5 B Gel Pre Run 11 5 C Sample Preparation and Loading 12 5 D Gel Electrophoresis 13 5 E Detection 13 6 Data Analysis 15 6 A Background Adjustment 15 6 B Color Separation Using Individual Bands 16 6 C Color Separation Using Bands In a Single Lane 16 6 D Autobanding
50. protocol The preferred protocols for use with the GeneAmp PCR system 9600 9700 and 2400 and Perkin Elmer model 480 thermal cyclers are provided below 3 After completion of the thermal cycling protocol store amplified samples at 20 C in a light protected box Note Long term storage of amplified samples at 4 C or higher may produce degradation products 8 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD016 Revised 7 15 www promega com Protocol for the GeneAmp PCR System 9700 Thermal Cycler1 Protocol for the GeneAmp PCR System 2400 Thermal Cycler 95 C for 11 minutes then 96 C for 1 minute then ramp 100 to 94 C for 30 seconds ramp 29 to 60 C for 30 seconds ramp 23 to 70 C for 45 seconds for 10 cycles then ramp 100 to 90 C for 30 seconds ramp 29 to 60 C for 30 seconds ramp 23 to 70 C for 45 seconds for 22 cycles then 60 C for 30 minutes 4 C soak 95 C for 11 minutes then 96 C for 1 minute then ramp 100 to 94 C for 30 seconds ramp 100 to 60 C for 30 seconds ramp 23 to 70 C for 45 seconds for 10 cycles then ramp 100 to 90 C for 30 seconds ramp 100 to 60 C for 30 seconds ramp 23 to 70 C for 45 seconds for 22 cycles then 60 C for 30 minutes 4 C soak Protocol for the GeneAmp PCR System 9600 Thermal Cycler Protocol for the Perkin Elmer Model 480 Th
51. robabilities Paternity Indices and Power of Exclusion of the PowerPlex 16 BIO System in Various Populations African American Caucasian American Hispanic American Asian American Matching Probability 1 in 1 41 1018 1 in 1 83 1017 1 in 2 93 1017 1 in 3 74 1017 Paternity Index 2 510 000 1 520 000 522 000 4 110 000 Power of Exclusion 0 9999996 0 9999994 0 9999983 0 9999998 32 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD016 Revised 7 15 www promega com 9 C DNA Extraction and Quantitation Methods and Automation Support The DNA IQ System Cat DC6700 is a DNA isolation system designed specifically for forensic and paternity samples 31 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 materi
52. rotocols for use of the PowerPlex 16 BIO System with the Perkin Elmer model 480 and GeneAmp PCR system 9600 9700 and 2400 thermal cyclers in addition to protocols for separation and detection of amplified materials Information about other Promega fluorescent STR systems is available upon request from Promega or online at www promega com geneticidentity Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 3 www promega com TMD016 Revised 7 15 2 Product Components and Storage Conditions P R O D U C T S I Z E C AT PowerPlex 16 BIO System 400 reactions DC6540 Not For Medical Diagnostic Use Cat DC6540 contains sufficient reagents for 400 reactions of 25 l each Includes Pre amplification Components Box Blue Label 4 300 l Gold STHR 10X Buffer 4 250 l PowerPlex 16 BIO 10X Primer Pair Mix 25 l 2800M Control DNA 10ng l Post amplification Components Box Beige Label 4 125 l PowerPlex 16 BIO Allelic Ladder Mix 100 l Matrix 16 BIO 2 300 l Internal Lane Standard ILS 600 BIO 2 1ml Bromophenol Blue Loading Solution 250 l Gel Tracking Dye The PowerPlex 16 BIO Allelic Ladder Mix is provided in a separate sealed bag for shipping This component should be moved to the post amplification box after opening Storage Conditions Store all components except the 2800M
53. rst possible 5 nucleotide of a repeat motif and 2 for STR loci not associated with a coding gene the first database entry or original literature description shall be used 2Amelogenin is not an STR but displays a 106 base X specific band and a 112 base Y specific band FL fluorescein JOE 6 carboxy 4 5 dichloro 2 7 dimethoxyfluorescein NA not applicable Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 29 www promega com TMD016 Revised 7 15 Table 7 The PowerPlex 16 BIO System Allelic Ladder Information STR Locus Label Size Range of Allelic Ladder Components1 2 bases Repeat Numbers of Allelic Ladder Components Repeat Numbers of Alleles Not Present in Allelic Ladder3 4 Penta E FL 379 474 5 24 20 3 D18S51 FL 290 366 8 10 10 2 11 13 13 2 14 27 D21S11 FL 203 259 24 24 2 25 25 2 26 28 28 2 29 29 2 30 30 2 31 31 2 32 32 2 33 33 2 34 34 2 35 35 2 36 38 TH01 FL 156 195 4 9 9 3 10 11 13 3 D3S1358 FL 115 147 12 20 FGA Rhodamine Red X 322 444 16 30 31 2 43 2 44 2 45 2 46 2 18 2 19 2 22 2 23 2 24 2 25 2 TPOX Rhodamine Red X 262 290 6 13 D8S1179 Rhodamine Red X 203 247 7 18 vWA Rhodamine Red X 123 171 10 22 Amelogenin5 Rhodamine Red X 106 112 X Y Penta D JOE
54. s a trademark of Molecular Probes Inc and Texas Red is a registered trademark of Molecular Probes Inc b U S Pat No 6 238 863 Chinese Pat No ZL99802696 4 European Pat No 1058727 Japanese Pat No 4494630 and other patents pending c Australian Pat No 724531 Canadian Pat No 2 251 793 Korean Pat No 290332 Singapore Pat No 57050 Japanese Pat Nos 3602142 and 4034293 Chinese Pat Nos ZL99813729 4 and ZL97194967 0 European Pat No 0960207 and other patents pending d The purchase of this product does not convey a license to use AmpliTaq Gold DNA polymerase You should purchase AmpliTaq Gold DNA polymerase licensed for the forensic and human identity field directly from your authorized enzyme supplier e Allele sequences for one or more of the loci vWA FGA D8S1179 D21S11 and D18S51 in allelic ladder mixtures is licensed under U S Pat Nos 7 087 380 7 645 580 Australia Pat No 2003200444 and corresponding patent claims outside the US 2001 2002 2007 2008 2011 2012 2015 Promega Corporation All Rights Reserved Maxwell Plexor and PowerPlex are registered trademarks of Promega Corporation Differex DNA IQ and Slicprep are trademarks of Promega Corporation AmpliTaq Gold and GeneAmp are registered trademarks of Roche Molecular Systems Inc ART is a registered trademark of Molecular Bio Products Inc FMBIO is a registered trademark of Hitachi Software Engineering Company Ltd FTA is a registered trad
55. tion 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 49 746 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 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 Vol 2 Greene Publishing Associates Inc and John Wiley and Sons 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 Press Cold Spring Harbor New York 7 PCR Technology Principles and Applications for DNA Amplification 1989 ed Erlich H A Stockton Press New York 8 PCR Protocols A Guide to Methods and Applications 1990 eds Innis M A et al Academic Press San Diego 9 Presley L A et al 1992 The implementation of the polymerase chain reaction PCR HLA DQ alpha typing by the FBI laboratory In The Third International Symposium on Human Identification 1992 Promega Corporation 245 69 10 Hartmann J M et
56. to 500 l of deionized water Bromophenol Blue Loading Solution 10mM NaOH 95 formamide 0 05 bromophenol blue Gel Tracking Dye 10mM NaOH 95 formamide 0 05 bromophenol blue 0 05 xylene cyanol FF Gold STHR 10X Buffer 500mM KCl 100mM Tris HCl pH 8 3 at 25 C 15mM MgCl2 1 Triton X 100 2mM each dNTP 1 6mg ml BSA TBE 10X buffer 107 8g Tris base 7 44g EDTA Na2EDTA 2H2O 55 0g boric acid Dissolve the Tris base and EDTA in 800ml of deionized water Slowly add the boric acid and monitor the pH until the desired pH of 8 3 is obtained Bring the volume to 1 liter with deionized water TE 4 buffer 10mM Tris HCl 0 1mM EDTA pH 8 0 1 21g Tris base 0 037g EDTA Na2EDTA 2H2O Dissolve Tris base and EDTA in 900ml of deionized water Adjust to pH 8 0 with HCl Bring the final volume to 1 liter with deionized water TE 4 buffer with 20 g ml glycogen 1 21g Tris base 0 037g EDTA Na2EDTA 2H2O 20 g ml glycogen Dissolve Tris base and EDTA in 900ml of deionized water Adjust to pH 8 0 with HCl Add glycogen Bring the final volume to 1 liter with deionized water Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 35 www promega com TMD016 Revised 7 15 9 G Related Products Product Size Cat PowerPlex 16 Monoplex System Penta E Fluorescein 100 reactions DC6591

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