Sample Protocols for Nucleic Acid Detection using MagPlex®TAG™ Microspheres Certain applications of xTAG microspheres, including those illustrated in this presentation, may be covered by patents owned by parties other than Luminex. Purchase of xTAG microspheres does not convey a license to any thirdparty patents unless explicitly stated in writing. You are responsible for conducting the necessary due diligence and securing rights to any thirdparty intellectual property required for your specific application(s) of xTAG® microspheres. Nothing herein is to be construed as recommending any practice or any product in violation of any patent or in violation of any law or regulation. 3/15/2010 ADV-004 1 PAGE Recommended Materials for MagPlex-TAG Microspheres 4 Source List for MagPlex-TAG Microspheres 4 Equipment for MagPlex-TAG Microspheres 5 Consumables for MagPlex-TAG Microspheres 6 Reagents for MagPlex-TAG Microspheres 7 xTAG Buffers 8 Sample Protocols for MagPlex-TAG Microspheres 9 Sample Protocol for Hybridization to MagPlex-TAG Microspheres – Washed Assay Format 9 Sample Protocol for Hybridization to MagPlex-TAG Microspheres – No Wash Protocol 11 Sample Protocol for Hybridization of Biotin-TAG Oligonucleotides to MagPlex-TAG 13 Microspheres Recommendations for xTAG ASPE Primer Design 15 Sample Protocol for Allele-Specific Primer Extension (ASPE) and Hybridization to MagPlex-TAG Microspheres – Washed Protocol 16 Sample Protocol for Allele-Specific Primer Extension (ASPE) and Hybridization to MagPlex-TAG Microspheres – No Wash Protocol 21 Recommendations for Optimization and Troubleshooting xTAG with ASPE Assays 25 Recommendations for xTAG OLA Probe Design 27 Sample Protocol for Oligonucleotide Ligation Assay (OLA) and Hybridization to MagPlex-TAG Microspheres – Washed Protocol 28 Sample Protocol for Oligonucleotide Ligation Assay (OLA) and Hybridization to 33 3/15/2010 ADV-004 2 MagPlex-TAG Microspheres – No Wash Protocol Recommendations for Optimization and Troubleshooting xTAG with OLA Assays 37 Recommendations for xTAG with PCR Primer Design 39 Sample Protocols for xTAG with PCR and Hybridization to MagPlex-TAG Microspheres 41 3/15/2010 ADV-004 3 SOURCE LIST for MagPlex-TAG Microspheres Company Applied Biosystems Bio-Rad BioTek Instruments, Inc. ColeParmer Corning (Costar) Dexter Magnetic Technologies Eppendorf Fisher Scientific Invitrogen GE Healthcare Life Sciences Millipore MOSS Inc. New England Biolabs PerkinElmer Rainin Roche Applied Science Sigma Qiagen Tecan Thermo Scientific (Pierce) VWR 3/15/2010 Phone Number Website See website www.appliedbiosystems.com 800-424-6723 www.bio-rad.com 888-451-5171 www.biotek.com 800-323-4340 800-492-1110 www.coleparmer.com www.corning.com/lifesciences See website www.dextermag.com 800-645-3050 800-766-7000 800-955-6288 www.eppendorfna.com www.fishersci.com www.invitrogen.com 800-526-3593 www.gelifesciences.com 800-645-5476 800-932-6677 www.millipore.com www.mosssubstrates.com 800-632-5227 www.neb.com 800-762-4000 800-472-4646 800-325-3010 800-426-8157 800-932-2687 www.perkinelmer.com www.rainin.com www.roche-appliedscience.com www.sigmaaldrich.com www.qiagen.com www.tecan.com 800-874-3723 www.piercenet.com 800-932-5000 www.vwr.com 800-262-1640 ADV-004 4 EQUIPMENT for MagPlex-TAG Microspheres Product Thermal cycler Source ABI, Bio-Rad, Eppendorf or Equivalent Catalog Number VWR 77888-200 VWR Fisher Eppendorf Fisher Eppendorf VWR Cole Parmer Rainin or Equivalent Millipore Millipore 53513-800 05-400-90 022622501 05-401-04 022638564 58816-121 08849-00 MAVM096OR WP6111560 Invitrogen 120-27 PerkinElmer (Customer Care) 5083175 Microcentrifuge Eppendorf 5415D with rotor or equivalent Centrifuge for 96-well plates (Eppendorf 5804) Microtiter plate rotor A-2-DWP (for Eppendorf 5804) Vortex Mixer Sonicator (mini) Pipettors P10, P20, P100, P1000, 8 ch. Vacuum Manifold Vacuum Pump Dynal MPC®-96S Magnetic Particle Concentrator 96 Well Plate Magnet 3/15/2010 ADV-004 various Various 5 CONSUMABLES for MagPlex-TAG Microspheres Product 0.2 mL PCR tubes - sterile 1.5 mL co-polymer microcentrifuge tubes 1.5 mL Eppendorf Protein LoBind microcentrifuge tubes 10 µL pipette tip refills 250 µL pipette tip refills 1000 µL pipette tip refills Thermowell 96-well P polycarbonate clear PCR plates - (recommended for XYP heater block) Microseal ‘A’ film 1.2µm PVDF filter microtiter plates (recommended for washed filter assays) 3/15/2010 Source Various Vendors USA Scientific Eppendorf Fisher Rainin Rainin Rainin Catalog Number ---1415-2500 022431081 13-698-794 GPS-10G GPS-250 GPS-1000 Costar Fisher 6509 07-200-541 Bio-Rad MSA-5001 Millipore MABVN1250 ADV-004 6 REAGENTS for MagPlex-TAG Microspheres Product Source Catalog Number Biotin-14-dCTP 19518-018 0.5 M EDTA, pH 8.0 Invitrogen New England Biolabs Invitrogen Roche Invitrogen 10297-018 11969064001 15575-020 HotStarTaq Master Mix Kit Qiagen 203443 HotStarTaq DNA Polymerase Qiagen New England Biolabs Sigma Invitrogen Sigma 203203 W4502 10977-015 S5150 Invitrogen S866 BSA dNTP Set Lambda Exonuclease Molecular Biology Grade Water 5 M Sodium Chloride Streptavidin-R-phycoerythrin (1 mg/ml) Streptavidin-PE conjugate with BSA Streptavidin-PE conjugate with BSA and Glycerol Taq DNA Ligase TE (Tris-EDTA) Buffer, pH 8.0, 100X 5M TMAC (Tetramethylammonium chloride solution) 1M Tris-HCL, pH 8.0 MOSS Inc. New England Biolabs Sigma  Triton X-100 3/15/2010 ADV-004 B9001S M0262L SAPE-001 SAPE-001G75 M0208S T9285 Sigma T3411 Sigma T3038 Sigma T8787 7 xTAG BUFFERS 2X Hybridization Buffer (also called 2X Tm Hybridization Buffer) Catalog Number Reagent 1M Tris-HCl, pH 8.0 5 M NaCl Triton X-100 Molecular Grade dH2O Sigma T3038 Sigma S5150 Sigma T8787 Sigma W4502 250 mL Amount/ Final Concentration 250 mL 0.2 M 0.4 M 0.16% --- 50 mL 20 mL 0.4 mL 179.6 mL Filter Sterilize and store at 4°°C 1.1X Hybridization Buffer (also called 1.1X Tm Hybridization Buffer) 250 mL Reagent Catalog Number Final Concentration Amount/ 250 mL 1M Tris-HCl, pH 8.0 5 M NaCl Triton X-100 Molecular Grade dH2O Sigma T3038 Sigma S5150 Sigma T8787 Sigma W4502 0.11 M 0.22M 0.088% --- 27.5 mL 11 mL 0.22 mL 211.28 mL Filter Sterilize and store at 4°°C 1X Hybridization Buffer (also called 1X Tm Hybridization Buffer) 250 mL Reagent Catalog Number Final Concentration Amount/ 250 mL 1M Tris-HCl, pH 8.0 5 M NaCl Triton X-100 Molecular Grade dH2O Sigma T3038 Sigma S5150 Sigma T8787 Sigma W4502 0.1 M 0.2M 0.08% --- 25 mL 10 mL 0.2 mL 214.8 mL Filter Sterilize and store at 4°°C Hybridization Buffer is also available from Luminex as a 10X concentrate: xTAG 10X Buffer (GR001A0058, GR001C0060, GR001D0095) 3/15/2010 ADV-004 8 SAMPLE PROTOCOL FOR HYBRIDIZATION TO MAGPLEX-TAG MICROSPHERES – WASHED ASSAY FORMAT MATERIALS • • • • • • • • Superparamagnetic carboxylated fluorescent microspheres with covalently attached anti-TAG sequences (MagPlex-TAG microspheres) Biotin-labeled targets with appropriate TAG sequence modification 2X Tm Hybridization Buffer – 0.4 M NaCl, 0.2 M Tris, 0.16% Triton X-100, pH 8.0 1X Tm Hybridization Buffer – 0.2 M NaCl, 0.1 M Tris, 0.08% Triton X-100, pH 8.0 Streptavidin-R-phycoerythrin (see REAGENTS list) 96 well V-bottom PCR plate & cover (see CONSUMABLES list) 96 well magnetic separator (see EQUIPMENT list) Pipettors, tips, microcentrifuge tubes, plates, etc. (see CONSUMABLES list) PROCEDURE Microspheres should be protected from prolonged exposure to light throughout this procedure. 1. Select the appropriate MagPlex-TAG microsphere sets and according to the instructions described in the Product Information Sheet provided with your microspheres. 2. Combine 2500 microspheres of each set per reaction. 3. Dilute/concentrate the MagPlex-TAG microsphere mixture to 100 of each microsphere set per µL in 2X Tm Hybridization Buffer by vortex and sonication for approximately 20 seconds. 4. Aliquot 25 µL of the MagPlex-TAG microsphere mixture to each well. 5. Add 25 µL of dH2O to each background well. 6. Add 5 to 25 µL of biotinylated, TAGged targets to the sample wells. (Note: For synthetic biotin-TAG oligonucleotide targets, use 5 to 200 femtomoles per reaction.) 7. Adjust the total volume to 50 µL by adding the appropriate volume of dH2O to each sample well. 8. Cover the plate to prevent evaporation and denature at 96°C for 90 seconds (optional). * 9. Hybridize at 37°C for 30 minutes. * 10. Pellet the MagPlex-TAG microspheres by placing the plate on a magnetic separator and allow separation to occur for 30 to 60 seconds. Remove the supernatant. See Technical Note. 3/15/2010 ADV-004 9 11. Resuspend the pelleted MagPlex-TAG microspheres in 75 µL of 1X Tm Hybridization Buffer. 12. Repeat steps 11 and 12. This is a total of two washes. 13. Pellet the MagPlex-TAG microspheres by placing the plate on a magnetic separator and allow separation to occur for 30 to 60 seconds. Remove the supernatant. 14. Resuspend the pelleted MagPlex-TAG microspheres in 75 µL of 1X Tm Hybridization Buffer containing 2-8 µg/mL streptavidin-R-phycoerythrin. 15. Incubate at 37°C for 15 minutes. 16. Analyze 50 µL at 37°C on the Luminex analyzer according to the system manual. * These steps can be performed on a thermal cycler programmed as follows – Hold at 96°°C, 90 seconds Hold at 37°°C, FOREVER Technical Note: Alternatively, wash steps can be performed by centrifugation or vacuum filtration. • Pellet the MagPlex-TAG microspheres by centrifugation at ≥ 2250 x g for 3 minutes and remove the supernatant. • Pre-wet a 1.2 µm Millipore filter plate with 1X Tm Hybridization Buffer and filter by vacuum manifold. Transfer the reactions to the pre-wetted filter plate and remove the supernatant by vacuum filtration. Wash twice with 100 µL 1X Tm Hybridization Buffer. Proceed with step 14. 3/15/2010 ADV-004 10 SAMPLE PROTOCOL FOR HYBRIDIZATION TO MAGPLEX-TAG MICROSPHERES – NO WASH PROTOCOL MATERIALS • • • • • • • Superparamagnetic carboxylated fluorescent microspheres with covalently attached anti-TAG sequences 1.1X Tm Hybridization Buffer – 0.22 M NaCl, 0.22 M Tris, 0.088% Triton X-100, pH 8.0 1X Tm Hybridization Buffer – 0.2 M NaCl, 0.1 M Tris, 0.08% Triton X-100, pH 8.0 Streptavidin-R-phycoerythrin (See REAGENTS list) 96 well V-bottom PCR plate and cover (See CONSUMABLES list) Pipettors, tips, microcentrifuge tubes, etc. (See CONSUMABLES list) DNA samples PROCEDURE Microspheres should be protected from prolonged exposure to light throughout this procedure. 1. Select the appropriate MagPlex-TAG microsphere sets and resuspend according to the instructions described in the Product Information Sheet provided with your microspheres. 2. Combine 2500 microspheres of each set per reaction. 3. Dilute/concentrate the MagPlex-TAG microsphere mixture to 111 of each microsphere set per µL in 1.1X Tm Hybridization Buffer and mix by vortex and sonication for approximately 20 seconds. 4. Aliquot 22.5 µL of the MagPlex-TAG microsphere mixture to each well. 5. Add 2.5 µL of dH2O to each background well. 6. Add 2.5 µL of each sample to the appropriate wells. 7. Cover the plate to prevent evaporation and denature at 96°C for 90 seconds (optional). * 8. Hybridize at 37°C for 30 minutes. * 9. Prepare Reporter Mix by diluting streptavidin-R-phycoerythrin to 10 µg/mL in 1X Tm Hybridization Buffer. 10. Add 100 µL Reporter Mix to each well. Mix gently. 3/15/2010 ADV-004 11 11. Incubate at 37°C for 15 minutes. 12. Analyze 100 µL at 37°C on the Luminex analyzer according to the system manual. * These steps can be performed on a thermal cycler programmed as follows – Hold at 96°°C, 90 seconds Hold at 37°°C, FOREVER 3/15/2010 ADV-004 12 SAMPLE PROTOCOL FOR HYBRIDIZATION OF BIOTIN-TAG OLIGONUCLEOTIDES TO MAGPLEX-TAG MICROSPHERES MATERIALS • Superparamagnetic carboxylated fluorescent microspheres with covalently attached anti-TAG sequences • Biotin-labeled targets with appropriate TAG sequence modification • 2X Tm Hybridization Buffer – 0.4 M NaCl, 0.2 M Tris, 0.16% Triton X-100, pH 8.0 • 1X Tm Hybridization Buffer – 0.2 M NaCl, 0.1 M Tris, 0.08% Triton X-100, pH 8.0 • Streptavidin-R-phycoerythrin (see REAGENTS list) • 96 well V-bottom PCR plate and cover (see CONSUMABLES list) • Pipettors, tips, microcentrifuge tubes, plates, etc. (see CONSUMABLES list) PROCEDURE Microspheres should be protected from prolonged exposure to light throughout this procedure. 1. Select the appropriate MagPlex-TAG microsphere sets and resuspend according to the instructions described in the Product Information Sheet provided with your microspheres. 2. Combine 2500 microspheres of each set per reaction. 3. Dilute/concentrate the MagPlex-TAG microsphere mixture to 100 of each microsphere set per µL in 2X Tm Hybridization Buffer by vortex and sonication for approximately 20 seconds. (Note: 25 µL are required for each reaction.) 4. Aliquot 25 µL of the MagPlex-TAG microsphere mixture to each well. 5. Add 25 µL of dH2O to each background well. 6. Add 5 to 25 µL of biotin-TAG oligonucleotides (5 to 200 femtomoles) to the sample wells. 7. Adjust the total volume to 50 µL by adding the appropriate volume of dH2O to each sample well. 8. Cover the plate to prevent evaporation and denature at 96°C for 90 seconds (optional). * 9. Hybridize at 37°C for 30 minutes. * 10. Dilute streptavidin-R-phycoerythrin to 10 µg/mL in 1X Tm Hybridization Buffer. (Note: 25 µL are required for each reaction.) 3/15/2010 ADV-004 13 11. Add 25 µL of 10 µg/mL streptavidin-R-phycoerythrin to each well and mix by gently pipetting up and down several times. (Note: Final concentration of streptavidin-Rphycoerythrin should be 2-4 µg/mL). 12. Incubate at 37°C for 15 minutes. 13. Analyze 50 µL at 37°C on the Luminex analyzer according to the system manual. * These steps can be performed on a thermal cycler programmed as follows – Hold at 96°°C, 90 seconds Hold at 37°°C, FOREVER 3/15/2010 ADV-004 14 Recommendations for xTAG ASPE Primer Design Oligonucleotide Synthesis PCR Primer Design 1. PCR primers should be designed to amplify a region containing the SNP of interest. 2. PCR primers should not be labeled. 3. Amplicon size is not restricted. ASPE Primer Design 1. ASPE primers should be synthesized for all sequence variants and should be from the same DNA strand (per target sequence). 2. ASPE primers should be matched for melting temperature at 51-56°C. 3. ASPE primers should extend out to and include the SNP as the 3’ nucleotide. 4. Use oligo design software to select an appropriate TAG sequence. 5. The primer is synthesized with the TAG sequence incorporated at the 5’ end. MagPlex-TAG Microsphere 3/15/2010 ADV-004 15 SAMPLE PROTOCOL FOR ALLELE-SPECIFIC PRIMER EXTENSION (ASPE) AND HYBRIDIZATION TO MAGPLEX-TAG MICROSPHERES – WASHED PROTOCOL MATERIALS • • • • • • • • • • • • • • • Superparamagnetic carboxylated fluorescent microspheres with covalently attached anti-TAG sequences PCR amplification primers for each target resuspended in sterile ddH2O. PCR primers are reconstituted to 1 mM (1 nanomole/µL). ASPE primers with 5’ TAG modification resuspended in sterile ddH2O. ASPE primers are reconstituted to 1 mM (1 nanomole/µL). Qiagen HotStarTaq 2X Master Mix (Qiagen, Cat. No. 203443) or equivalent ExoSAP-IT (GE Healthcare, Cat. No. US78200), (or separate Exo I and SAP), or equivalent Platinum Tsp DNA polymerase, ASPE 10X Buffer, 50 mM MgCl2 (Invitrogen, Cat. No. 11448-024) or equivalent dNTPs at 100 mM each (Invitrogen, Cat. No. 10297-018) or equivalent Biotin-14-dCTP at 0.4 mM (Invitrogen, Cat. No. 19518-018) or equivalent 2X Tm Hybridization Buffer – 0.4 M NaCl, 0.2 M Tris, 0.16% Triton X-100, pH 8.0 1X Tm Hybridization Buffer – 0.2 M NaCl, 0.1 M Tris, 0.08% Triton X-100, pH 8.0 Streptavidin-R-phycoerythrin (See REAGENTS list) 96 well V-bottom PCR plate and cover (See CONSUMABLES list) 96 well plate magnetic separator (See EQUIPMENT list) Pipettors, tips, microcentrifuge tubes, etc. (See CONSUMABLES list) Genomic DNA samples 3/15/2010 ADV-004 16 PROCEDURES Multiplexed PCR Reaction – PCR should be performed under optimized conditions. The parameters listed below are for example purposes only. Each final reaction contains: 1X Qiagen PCR reaction buffer 1.5 mM MgCl2 200 µM each dNTP 0.2 µM each primer 2.5 Units Qiagen HotStarTaq polymerase 50 ng template PCR Cycling Parameters: HOLD: 95°C, 15 minutes (for enzyme activation) CYCLE: 94°C, 30 seconds 55°C, 30 seconds 72°C, 30 seconds 35 CYCLES HOLD: 72°C, 7 minutes HOLD: 4°C, FOREVER EXO/SAP Treatment Treat 7.5 µL of each PCR reaction with ExoSAP-IT according to the following procedure: PCR reaction ExoSAP-IT 7.5 µL 3.0 µL 10.5 µL The following steps should be performed on a thermal cycler: Mix and incubate at 37°C for 30 minutes. Inactivate ExoSAP-IT by heating to 80°C for 15 minutes. Hold the treated reactions at 4°C. 3/15/2010 ADV-004 17 Multiplex ASPE Reaction Each 20 µL final reaction contains: 1X ASPE Buffer (20 mM Tris-HCl, pH 8.4; 50 mM KCl) 1.25 mM MgCl2 25 nM each TAG-ASPE primer 0.75 U Tsp DNA polymerase 5 µM dATP, dTTP, dGTP 5 µM biotin-dCTP 5 µL treated PCR reaction dH2O (to 20 µL) 2X ASPE Master Mix (10 µL/reaction): 10X ASPE reaction buffer 50 mM MgCl2 20X TAG-ASPE primer mix (500 nM each) 5 U/µL Tsp DNA polymerase 20X dNTP mix (-dCTP) (100 µM each) 2 µL 0.5 µL 1 µL (dilute 1 mM stocks 1:2000 for 20X mix) 0.15 µL 1 µL (dilute 100 mM stocks 1:1000 for 20X mix) 400 µM biotin-dCTP dH2O 0.25 µL 5.1 µL 10 µL ASPE Cycling Parameters: HOLD: 96°C, 2 minutes CYCLE: 94°C, 30 seconds 55°C, 1 minute 74°C, 2 minutes 30 CYCLES HOLD: 3/15/2010 4°C, FOREVER ADV-004 18 Hybridization to MagPlex-TAG Microspheres Microspheres should be protected from prolonged exposure to light throughout this procedure. 1. Select the appropriate MagPlex-TAG microsphere sets and resuspend according to the instructions described in the Product Information Sheet provided with your microspheres. 2. Combine 2500 microspheres of each set per reaction. 3. Dilute/concentrate the MagPlex-TAG microsphere mixture to 100 of each microsphere set per µL in 2X Tm Hybridization Buffer and mix by vortex and sonication for approximately 20 seconds. 4. Aliquot 25 µL of the MagPlex-TAG microsphere mixture to each well. 5. Add 25 µL of dH2O to each background well. 6. Add 5 to 25 µL of each ASPE reaction to appropriate wells. (Note: 1-5 µL is usually sufficient.) 7. Adjust the total volume to 50 µL by adding the appropriate volume of dH2O to each sample well. 8. Cover the plate to prevent evaporation and denature at 96°C for 90 seconds. * 9. Hybridize at 37°C for 30 minutes. * 10. Pellet the MagPlex-TAG microspheres by placing the plate on a magnetic separator and allow separation to occur for 30 to 60 seconds. Remove the supernatant. See Technical Note. 11. Resuspend the pelleted MagPlex-TAG microspheres in 75 µL of 1X Tm Hybridization Buffer. 12. Repeat steps 11 and 12. This is a total of two washes. 13. Pellet the MagPlex-TAG microspheres by placing the plate on a magnetic separator and allow separation to occur for 30 to 60 seconds. Remove the supernatant. 14. Resuspend microspheres in 75 µL of 1X Tm Hybridization Buffer containing 2-8 µg/mL streptavidin-R-phycoerythrin. 15. Incubate at 37°C for 15 minutes. 16. Analyze 50 µL at 37°C on the Luminex analyzer according to the system manual. * These steps can be performed on a thermal cycler programmed as follows – Hold at 96°°C, 90 seconds Hold at 37°°C, FOREVER 3/15/2010 ADV-004 19 Technical Note: Alternatively, wash steps can be performed by centrifugation or vacuum filtration. • Pellet the MagPlex-TAG microspheres by centrifugation at ≥ 2250 x g for 3 minutes and remove the supernatant. • Pre-wet a 1.2 µm Millipore filter plate with 1X Tm Hybridization Buffer and filter by vacuum manifold. Transfer the reactions to the pre-wetted filter plate and remove the supernatant by vacuum filtration. Wash twice with 100 µL 1X Tm Hybridization Buffer. Proceed with step 14. 3/15/2010 ADV-004 20 SAMPLE PROTOCOL FOR ALLELE-SPECIFIC PRIMER EXTENSION (ASPE) AND HYBRIDIZATION TO MAGPLEX-TAG MICROSPHERES – NO WASH PROTOCOL MATERIALS • • • • • • • • • • • • • • Superparamagnetic carboxylated fluorescent microspheres with covalently attached xTAG anti-TAG sequences PCR amplification primers for each target resuspended in sterile ddH2O. PCR primers are reconstituted to 1 mM (1 nanomole/µL). ASPE primers with 5’ TAG modification resuspended in sterile ddH2O. ASPE primers are reconstituted to 1 mM (1 nanomole/µL). Qiagen HotStarTaq 2X Master Mix (Qiagen, Cat. No. 203443) or equivalent ExoSAP-IT (GE Healthcare, Cat. No. US78200), (or separate Exo I and SAP), or equivalent Platinum Tsp DNA polymerase, ASPE 10X Buffer, 50 mM MgCl2 (Invitrogen, Cat. No. 11448-024) or equivalent dNTPs at 100 mM each (Invitrogen, Cat. No. 10297-018) or equivalent Biotin-14-dCTP at 0.4 mM (Invitrogen, Cat. No. 19518-018) or equivalent 1.1X Tm Hybridization Buffer – 0.22 M NaCl, 0.22 M Tris, 0.088% Triton X-100, pH 8.0 1X Tm Hybridization Buffer – 0.2 M NaCl, 0.1 M Tris, 0.08% Triton X-100, pH 8.0 Streptavidin-R-phycoerythrin (See REAGENTS list) 96 well V-bottom PCR plate and cover (See CONSUMABLES list) Pipettors, tips, microcentrifuge tubes, etc. (See CONSUMABLES list) genomic DNA samples 3/15/2010 ADV-004 21 PROCEDURES Multiplexed PCR Reaction – PCR should be performed under optimized conditions. The parameters listed below are for example purposes only. Each final reaction contains: 1X Qiagen PCR reaction buffer 1.5 mM MgCl2 200 µM each dNTP 0.2 µM each primer 2.5 Units Qiagen HotStarTaq polymerase 50 ng template PCR Cycling Parameters: HOLD: 95°C, 15 minutes (for enzyme activation) CYCLE: 94°C, 30 seconds 55°C, 30 seconds 72°C, 30 seconds 35 CYCLES HOLD: 72°C, 7 minutes HOLD: 4°C, FOREVER EXO/SAP Treatment Treat 7.5 µL of each PCR reaction with ExoSAP-IT according to the following procedure: PCR reaction ExoSAP-IT 7.5 µL 3.0 µL 10.5 µL The following steps should be performed on a thermal cycler: Mix and incubate at 37°C for 30 minutes. Inactivate ExoSAP-IT by heating to 80°C for 15 minutes. Hold the treated reactions at 4°C. 3/15/2010 ADV-004 22 Multiplex ASPE Reaction Each 20 µL final reaction contains: 1X ASPE Buffer (20 mM Tris-HCl, pH 8.4; 50 mM KCl) 1.25 mM MgCl2 25 nM each TAG-ASPE primer 0.75 Platinum Tsp DNA polymerase 5 µM dATP, dTTP, dGTP 5 µM biotin-dCTP 5 µL treated PCR reaction dH2O (to 20 µL) 2X ASPE Master Mix (10 µL/reaction): 10X ASPE reaction buffer 50 mM MgCl2 20X TAG-ASPE primer mix (500 nM each) 2 µL 0.5 µL 1 µL (dilute 1 mM stocks 1:2000 for 20X mix) 5 U/µL Tsp DNA polymerase 20X dNTP mix (-dCTP) (100 µM each) 0.15 µL 1 µL (dilute 100 mM stocks 1:1000 for 20X mix) 400 µM biotin-dCTP dH2O 0.25 µL 5.1 µL 10 µL ASPE Cycling Parameters: HOLD: 96°C, 2 minutes CYCLE: 94°C, 30 seconds 55°C, 1 minute 74°C, 2 minutes 30 CYCLES HOLD: 3/15/2010 4°C, FOREVER ADV-004 23 Hybridization to MagPlex-TAG Microspheres – No Wash Protocol Microspheres should be protected from prolonged exposure to light throughout this procedure. 1. Select the appropriate MagPlex-TAG microsphere sets and resuspend according to the instructions described in the Product Information Sheet provided with your microspheres. 2. Combine 2500 microspheres of each set per reaction. 3. Dilute/concentrate the MagPlex-TAG microsphere mixture to 111 of each microsphere set per µL in 1.1X Tm Hybridization Buffer by vortex and sonication for approximately 20 seconds. 4. Aliquot 22.5 µL of the MagPlex-TAG microsphere mixture to each well. 5. Add 2.5 µL of dH2O to each background well. 6. Add 2.5 µL of each ASPE reaction to appropriate wells. 7. Cover the plate to prevent evaporation and denature at 96°C for 90 seconds. * 8. Hybridize at 37°C for 30 minutes. * 9. Prepare Reporter Mix by diluting streptavidin-R-phycoerythrin to 10 µg/mL in 1X Tm Hybridization Buffer. 10. Add 100 µL to each well. Mix gently. 11. Incubate at 37°C for 15 minutes. 12. Analyze 100 µL at 37°C on the Luminex analyzer according to the system manual. * These steps can be performed on a thermal cycler programmed as follows – Hold at 96°°C, 90 seconds Hold at 37°°C, FOREVER 3/15/2010 ADV-004 24 Recommendations for Optimization and Troubleshooting xTAG with ASPE Assays Low Reporter Intensity 1. Verify the hybridization assay by direct hybridization to 5 and 50 femtomoles of labeled oligonucleotide targets (i.e., biotinylated TAGs). 2. Titrate the target input to determine the optimal amount for hybridization. 3. Verify the production of the PCR products (ASPE templates). 4. Titrate the template input to determine the optimal amount for ASPE. 5. Titrate the biotinylated dCTP input to determine the optimal concentration for ASPE. 6. Increase the number of cycles in the ASPE reaction. 7. Decrease and/or increase the ASPE annealing temperature. 8. Check the primer and template sequences for potential secondary structure. 9. Redesign the PCR primers. 10. Redesign the ASPE primers for the opposite DNA strand. 11. Lengthen the ASPE primers. Poor Discrimination 1. Increase the ASPE annealing temperature. 2. Redesign the ASPE primers for the opposite DNA strand. 3. Shorten the “leaky” ASPE primer. Poor Reporter Distribution Between Alleles 1. Redesign the ASPE primers for the opposite DNA strand. 2. Lengthen the ASPE primer to increase signal on the “low” allele. 3. Shorten the ASPE primer to decrease signal on the “high” allele. 3/15/2010 ADV-004 25 High Background 1. If high background is observed for the PCR negative control, verify performance of the Exo/SAP step. 2. If the high background is due to contamination of the PCR reaction, replace the PCR reagents. 3. If high background is observed for the hybridization negative control, replace the hybridization reagents. 4. If high background is observed for the ASPE negative control, replace the ASPE reagents. 3/15/2010 ADV-004 26 Recommendations for xTAG OLA Probe Design Oligonucleotide Synthesis PCR Primer Design 1. PCR primers should be designed to amplify a region containing the SNP of interest. 2. PCR primers should not be labeled. 3. Amplicon size is not restricted. OLA Probe Design 1. OLA probes should be synthesized for all sequence variants and should be from the same DNA strand (per target sequence). 2. OLA probes should be matched for melting temperature at 51-56°C. 3. OLA probes should extend out to and include the SNP as the 3’ nucleotide. 4. Use oligo design software to select an appropriate TAG sequence. 5. The OLA probe is synthesized with the TAG sequence incorporated at the 5’ end. 6. The reporter probe should have a melting temperature of 51-56°C. 7. The reporter probe should begin with the nucleotide immediately downstream from the SNP as the 5’ nucleotide. 8. The reporter probe must be modified with phosphate at the 5’ end and with biotin at the 3’ end. MagPlexTAG 3/15/2010 ADV-004 27 SAMPLE PROTOCOL FOR OLIGONUCLEOTIDE LIGATION ASSAY (OLA) AND HYBRIDIZATION TO MAGPLEX-TAG MICROSPHERES – WASHED PROTOCOL MATERIALS • • • • • • • • • • • • • Superparamagnetic carboxylated fluorescent microspheres with covalently attached anti-TAG sequences PCR amplification primers for each target resuspended in sterile ddH2O. PCR primers are reconstituted to 1 mM (1 nanomole/µL). OLA probes with 5’ TAG modification resuspended in sterile ddH2O. OLA probes are reconstituted to 1 mM (1 nanomole/µL). Reporter probes with 5’ phosphate and 3’ biotin modifications resuspended in sterile ddH2O. Reporter probes are reconstituted to 1 mM (1 nanomole/µL). Qiagen HotStarTaq 2X Master Mix (Qiagen, Cat. No. 203443) or equivalent Taq DNA Ligase, 10X Taq DNA Ligase Buffer (New England Biolabs, Cat. No. M0208S) or equivalent 2X Tm Hybridization Buffer – 0.4 M NaCl, 0.2 M Tris, 0.16% Triton X-100, pH 8.0 1X Tm Hybridization Buffer – 0.2 M NaCl, 0.1 M Tris, 0.08% Triton X-100, pH 8.0 Streptavidin-R-phycoerythrin (See REAGENTS list) 96 well V-bottom PCR plate & cover (See CONSUMABLES list) 96 well magnetic plate separator (See EQUIPMENT list) Pipettors, tips, microcentrifuge tubes, etc. (See CONSUMABLES list) Genomic DNA samples 3/15/2010 ADV-004 28 PROCEDURES Multiplexed PCR Reaction – PCR should be performed under optimized conditions. The parameters listed below are for example purposes only. Each final reaction contains: 1X Qiagen PCR reaction buffer 1.5 mM MgCl2 200 µM each dNTP 0.2 µM each primer 2.5 Units Qiagen HotStarTaq polymerase 50 ng template PCR Cycling Parameters: HOLD: 95°C, 15 minutes (for enzyme activation) CYCLE: 94°C, 30 seconds 55°C, 30 seconds 72°C, 30 seconds 35 CYCLES HOLD: 72°C, 7 minutes HOLD: 4°C, FOREVER 3/15/2010 ADV-004 29 Multiplex OLA Reaction Each 20 µL final reaction contains: 1X Taq DNA Ligase Buffer (20 mM Tris-HCl, 25 mM potassium acetate, 10 mM magnesium acetate, 10 mM dithiothreitol, 1 mM NAD, 0.1% Triton X-100, pH 7.6) 10 U Taq DNA Ligase 5 nM each TAG-OLA probe 250 nM each Reporter probe 3 to 20 ng PCR target (usually 0.5 to 5 µL) dH2O (to 20 µL) 2X OLA Master Mix (10µL/reaction) 10X Taq DNA Ligase Buffer2µL Taq DNA Ligase (40,000 U/mL) 20X TAG-OLA probe mix (100 nM each) 20X mix) 20X Reporter probe mix (5 µM each) mix) dH2O 0.25 µL 1 µL (dilute 1 mM stocks 1:10,000 for 1 µL (dilute 1 mM stocks 1:200 for 20X 5.75 µL 10 µL OLA Cycling Parameters: HOLD: 96°C, 2 minutes CYCLE: 94°C, 15 seconds 37°C, 1 minute 30 CYCLES HOLD: 3/15/2010 4°C, FOREVER ADV-004 30 Hybridization to MagPlex-TAG Microspheres Microspheres should be protected from prolonged exposure to light throughout this procedure. 1. Select the appropriate MagPlex-TAG microsphere sets and resuspend according to the instructions described in the Product Information Sheet provided with your microspheres. 2. Combine 2500 microspheres of each set per reaction. 3. Dilute/concentrate the MagPlex-TAG microsphere mixture to 100 of each microsphere set per µL in 2X Tm Hybridization Buffer by vortex and sonication for approximately 20 seconds. 4. Aliquot 25 µL of the MagPlex-TAG microsphere mixture to each well. 5. Add 25 µL of dH2O to each background well. 6. Add 5 to 25 µL of each OLA reaction to the appropriate wells. (Note: 5 µL is usually sufficient.) 7. Adjust the total volume to 50 µL by adding the appropriate volume of dH2O to each sample well. 8. Cover the plate to prevent evaporation and denature at 96°C for 90 seconds. * 9. Hybridize at 37°C for 30 minutes. * 10. Pellet the MagPlex-TAG microspheres by placing the plate on a magnetic separator and allow separation to occur for 30 to 60 seconds. Remove the supernatant. See Technical Note. 11. Resuspend the pelleted MagPlex TAG microspheres in 75 µL of 1X Tm Hybridization Buffer. 12. Repeat steps 11 and 12. This is a total of two washes. 13. Pellet the MagPlex-TAG microspheres by placing the plate on a magnetic separator and allow separation to occur for 30 to 60 seconds. Remove the supernatant. 14. Resuspend microspheres in 75 µL of 1X Tm Hybridization Buffer containing 2-8 µg/mL streptavidin-R-phycoerythrin. 15. Incubate at 37°C for 15 minutes. 16. Analyze 50 µL at 37°C on the Luminex analyzer according to the system manual. * These steps can be performed on a thermal cycler programmed as follows – Hold at 96°°C, 90 seconds Hold at 37°°C, FOREVER 3/15/2010 ADV-004 31 Technical Note: Alternatively, wash steps can be performed by centrifugation or vacuum filtration. • Pellet the MagPlex-TAG microspheres by centrifugation at ≥ 2250 x g for 3 minutes and remove the supernatant. • Pre-wet a 1.2 µm Millipore filter plate with 1X Tm Hybridization Buffer and filter by vacuum manifold. Transfer the reactions to the pre-wetted filter plate and remove the supernatant by vacuum filtration. Wash twice with 100 µL 1X Tm Hybridization Buffer. Proceed with step 14. 3/15/2010 ADV-004 32 SAMPLE PROTOCOL FOR OLIGONUCLEOTIDE LIGATION ASSAY (OLA) AND HYBRIDIZATION TO MAGPLEX-TAG MICROSPHERES – NO WASH PROTOCOL MATERIALS • • • • • • • • • • • • Superparamagnetic carboxylated fluorescent microspheres with covalently attached xTAG anti-TAG sequences PCR amplification primers for each target resuspended in sterile ddH2O. PCR primers are reconstituted to 1 mM (1 nanomole/µL). OLA probes with 5’ TAG modification resuspended in sterile ddH2O. OLA probes are reconstituted to 1 mM (1 nanomole/µL). Reporter probes with 5’ phosphate and 3’ biotin modifications resuspended in sterile ddH2O. Reporter probes are reconstituted to 1 mM (1 nanomole/µL). Qiagen HotStarTaq 2X Master Mix (Qiagen, Cat. No. 203443) or equivalent Taq DNA Ligase, 10X Taq DNA Ligase Buffer (New England Biolabs, Cat. No. M0208S) or equivalent 1.1X Tm Hybridization Buffer – 0.22 M NaCl, 0.22 M Tris, 0.088% Triton X-100, pH 8.0 1X Tm Hybridization Buffer – 0.2 M NaCl, 0.1 M Tris, 0.08% Triton X-100, pH 8.0 Streptavidin-R-phycoerythrin (See REAGENTS list) 96 well V-bottom PCR plate & cover (See CONSUMABLES list) Pipettors, tips, microcentrifuge tubes, etc. (See CONSUMABLES list) Genomic DNA samples 3/15/2010 ADV-004 33 PROCEDURES Multiplexed PCR Reaction – PCR should be performed under optimized conditions. The parameters listed below are for example purposes only. Each final reaction contains: 1X Qiagen PCR reaction buffer 1.5 mM MgCl2 200 µM each dNTP 0.2 µM each primer 2.5 Units Qiagen HotStarTaq polymerase 50 ng template PCR Cycling Parameters: HOLD: 95°C, 15 minutes (for enzyme activation) CYCLE: 94°C, 30 seconds 55°C, 30 seconds 72°C, 30 seconds 35 CYCLES HOLD: 72°C, 7 minutes HOLD: 4°C, FOREVER 3/15/2010 ADV-004 34 Multiplex OLA Reaction Each 20 µL final reaction contains: 1X Taq DNA Ligase Buffer (20 mM Tris-HCl, 25 mM potassium acetate, 10 mM magnesium acetate, 10 mM dithiothreitol, 1 mM NAD, 0.1% Triton X-100, pH 7.6) 10 U Taq DNA Ligase 5 nM each TAG-OLA probe 250 nM each Reporter probe 3 to 20 ng PCR target (usually 0.5 to 5 µL) dH2O (to 20 µL) 2X OLA Master Mix (10µL/reaction) 10X Taq DNA Ligase Buffer Taq DNA Ligase (40,000 U/mL) 20X TAG-OLA probe mix (100 nM each) 2 µL 0.25 µL 1 µL (dilute 1 mM stocks 1:10,000 for 20X mix) 20X Reporter probe mix (5 µM each) 1 µL (dilute 1 mM stocks 1:200 for 20X mix) dH2O 5.75 µL 10 µL OLA Cycling Parameters: HOLD: 96°C, 2 minutes CYCLE: 94°C, 15 seconds 37°C, 1 minute 30 CYCLES HOLD: 3/15/2010 4°C, FOREVER ADV-004 35 Hybridization to MagPlex-TAG Microspheres – No Wash Protocol Microspheres should be protected from prolonged exposure to light throughout this procedure. 1. Select the appropriate MagPlex-TAG microsphere sets and resuspend according to the instructions described in the Product Information Sheet provided with your microspheres. 2. Combine 2500 microspheres of each set per reaction. 3. Dilute/concentrate the MagPlex-TAG microsphere mixture to 111 of each microsphere set per µL in 1.1X Tm Hybridization Buffer by vortex and sonication for approximately 20 seconds. 4. Aliquot 22.5 µL of the MagPlex-TAG microsphere mixture to each well. 5. Add 2.5 µL of dH2O to each background well. 6. Add 2.5 µL of each OLA reaction to the appropriate wells. 7. Cover the plate to prevent evaporation and denature at 96°C for 90 seconds. * 8. Hybridize at 37°C for 30 minutes. * 9. Prepare Reporter Mix by diluting SA-PE to 10 µg/mL in 1X Tm Buffer. 10. Add 100 µL Reporter Mix to each well. Mix gently. 11. Incubate at 37°C for 15 minutes. 12. Analyze 100 µL at 37°C on the Luminex analyzer according to the system manual. * These steps can be performed on a thermal cycler programmed as follows – Hold at 96°°C, 90 seconds Hold at 37°°C, FOREVER 3/15/2010 ADV-004 36 Recommendations for Optimization and Troubleshooting xTAG with OLA Assays Low Reporter Intensity 1. Verify the hybridization assay by direct hybridization to 5 and 50 femtomoles of labeled oligonucleotide targets (i.e., biotinylated TAGs). 2. Titrate the target input to determine the optimal amount for hybridization. 3. Verify the production of the PCR products (OLA templates). 4. Titrate the template input to determine the optimal amount for OLA. 5. Titrate the allele-specific and reporter probe inputs to determine optimal concentrations for OLA (Note: An allele-specific to reporter probe ratio of 1:50 improves the probability that an allele-specific probe will anneal adjacent to a reporter probe.) 6. Increase the number of cycles in the OLA reaction. 7. Decrease and/or increase the OLA annealing temperature. 8. Check the primer and template sequences for potential secondary structure. 9. Redesign the PCR primers. 10. Redesign the OLA primers for the opposite DNA strand. 11. Lengthen the OLA primers. Poor Discrimination 1. Increase the OLA annealing temperature. 2. Redesign the OLA probes for the opposite DNA strand. 3. Shorten the “leaky” OLA probe. Poor Reporter Distribution Between Alleles 1. Redesign the OLA probes for the opposite DNA strand. 2. Lengthen the OLA probes to increase signal on the “low” allele. 3. Shorten the OLA probes to decrease signal on the “high” allele. 3/15/2010 ADV-004 37 High Background 1. If the high background is due to contamination of the PCR reaction, replace the PCR reagents. 2. If high background is observed for the hybridization negative control, replace the hybridization reagents. 3. If high background is observed for the OLA negative control, replace the OLA reagents. 3/15/2010 ADV-004 38 Recommendations for xTAG with PCR Primer Design Oligonucleotide Synthesis PCR Reaction Strategy The PCR reaction should be designed so that the production of the anti-TAG sequence (complement of TAG) on the non-target strand is prevented or minimized. Try one of the following PCR amplification strategies: METHOD 1 – Asymmetric PCR – The TAGged primers (for the target strands) are in excess relative to the primers without TAG for the non-target strands. Optimize the ratio of TAGged to non-TAGged primers (usually 1:10 to 1:100). Include a biotinylated dNTP in the PCR reaction to biotinylate the TAGged target strand. METHOD 2 – PCR with Lambda Exonuclease Treatment – The primers without TAG are 5’ phosphorylated. The completed PCR reactions are treated with Lambda Exonuclease to degrade the phosphorylated non-target strands. Include a biotinylated dNTP in the PCR reaction to biotinylate the TAGged target strands. METHOD 3 – Spacer-modified TAGged Primers – Design the TAGged primers so that there is a spacer modification between the TAG and target-specific sequence to prevent amplification of the anti-TAG sequence in the non-target strand. The primers without TAG are 5’ biotinylated to label the PCR products. DO NOT DENATURE THE PCR REACTIONS PRIOR TO HYBRIDIZATION. PCR Primer Design 1. PCR primers should be designed to amplify a region containing the sequence of interest. 2. The discriminating target-specific PCR primers should be synthesized for all sequence variants and should be from the same DNA strand (per target sequence). 3. PCR primers should be matched for melting temperature at 51-56°C. 4. The target-specific PCR primer should extend out to and include the SNP or target-specific nucleotide as the 3’ nucleotide. 3/15/2010 ADV-004 39 5. Use oligo design software to select an appropriate TAG sequence. 6. The target-specific primer is synthesized with the TAG sequence incorporated at the 5’ end. 7. The primers for the non-target strands are designed according to the PCR strategy employed: METHOD 1 Asymmetric PCR – The primer without TAG is unmodified. METHOD 2 PCR with Lambda Exonuclease treatment – The primer without TAG is 5’ phosphorylated. METHOD 3 Spacer-modified TAGged primers – The primer without TAG is 5’ biotinylated METHODS 1 & 2 MagPlex-TAG Microsphere METHOD 3 MagPlex-TAG Microsphere 3/15/2010 ADV-004 40 SAMPLE PROTOCOLS FOR xTAG WITH PCR AND HYBRIDIZATION TO MAGPLEXTAG MICROSPHERES MATERIALS • • • • • • • • • • • • Superparamagnetic carboxylated fluorescent microspheres with covalently attached xTAG anti-TAG sequences PCR amplification primers for each target resuspended in sterile ddH2O. PCR primers are reconstituted to 1 mM (1 nanomole/µL). For each target, one primer has a unique TAG sequence, or a unique TAG and spacer (see METHOD 3), at the 5’ end upstream from the target-specific sequence. The other primer is designed according to one of the following methods: METHOD 1 Asymmetric PCR – primer without TAG is unmodified METHOD 2 PCR with Lambda Exonuclease treatment – primer without TAG is 5’ phosphorylated METHOD 3 Spacer-modified TAGged primers – primer without TAG is 5’ biotinylated Qiagen HotStarTaq Polymerase, 10X PCR Buffer, 25 mM MgCl2 (Qiagen, Cat. No. 203203) or equivalent Lambda Exonuclease and 10X reaction buffer (New England Biolabs, Cat. No. M0262L) or equivalent (for METHOD 2) dNTPs at 100 mM each (Invitrogen, Cat. No. 10297-018) or equivalent Biotin-14-dCTP at 0.4 mM (Invitrogen, Cat. No. 19518-018) or equivalent (for METHODS 1 and 2) 1.1X Tm Hybridization Buffer – 0.22 M NaCl, 0.22 M Tris, 0.088% Triton X-100, pH 8.0 1X Tm Hybridization Buffer – 0.2 M NaCl, 0.1 M Tris, 0.08% Triton X-100, pH 8.0 Streptavidin-R-phycoerythrin (See REAGENTS list) 96 well V-bottom PCR plate and cover (See CONSUMABLES list) Pipettors, tips, microcentrifuge tubes, etc. (See CONSUMABLES list) genomic DNA samples 3/15/2010 ADV-004 41 PROCEDURES Multiplexed PCR Reaction – PCR should be performed under optimized conditions. The parameters listed below are for example purposes only. METHOD 1 – Asymmetric PCR Note: Ratio of TAGged (excess) to non-TAGged (limiting) primer should be optimized in the 1:10 to 1:100 range. Ratio of biotinylated to unlabeled dNTPs may also need optimization. Each final reaction contains: 1X Qiagen PCR reaction buffer 1.5 mM MgCl2 200 µM each dNTP (-dCTP) 200 µM biotin-dCTP 0.4-1 µM each TAGged primer 0.004-0.1 µM each primer without TAG 2.5 Units Qiagen HotStarTaq polymerase 50 ng template METHOD 2 – PCR with Lambda Exonuclease Treatment Note: Ratio of biotinylated to unlabeled dNTPs may also need optimization. Each final reaction contains: 1X Qiagen PCR reaction buffer 1.5 mM MgCl2 200 µM each dNTP (-dCTP) 200 µM biotin-dCTP 0.2 µM each primer 2.5 Units Qiagen HotStarTaq polymerase 50 ng template METHOD 3 – Spacer-modified TAGged Primers Each final reaction contains: 1X Qiagen PCR reaction buffer 1.5 mM MgCl2 200 µM each dNTP 0.2 µM each primer 2.5 Units Qiagen HotStarTaq polymerase 50 ng template 3/15/2010 ADV-004 42 PCR Cycling Parameters: HOLD: 95°C, 15 minutes (for enzyme activation) CYCLE: 94°C, 30 seconds 55°C, 30 seconds 72°C, 30 seconds 35 CYCLES HOLD: 72°C, 7 minutes HOLD: 4°C, FOREVER Lambda Exonuclease Treatment – for METHOD 2 Treat each PCR reaction with Lambda Exonuclease according to the following sample protocol: PCR reaction 10X Lambda Exonuclease reaction buffer Lambda Exonuclease dH2O 5 µL 1 µL 5-10 Units to 10 µL Total Volume The following steps should be performed on a thermal cycler: Mix and incubate at 37°C for 30 minutes. Inactivate Lambda Exonuclease by heating to 80°C for 15 minutes. Hold the treated reactions at 4°C. 3/15/2010 ADV-004 43 Hybridization to MagPlex-TAG Microspheres – No Wash Protocol for METHODS 1 &2 See following No Wash Protocol for METHOD 3 Note: Washed Protocol may be necessary if using METHOD 1 or 2. See following Washed Protocol. Microspheres should be protected from prolonged exposure to light throughout this procedure. 1. Select the appropriate MagPlex-TAG microsphere sets and resuspend according to the instructions described in the Product Information Sheet provided with your microspheres. 2. Combine 2500 microspheres of each set per reaction. 3. Dilute/concentrate the MagPlex-TAG microsphere mixture to 125 of each microsphere set per µL in 1.1X Tm Hybridization Buffer by vortex and sonication for approximately 20 seconds. 4. Aliquot 20 µL of the MagPlex-TAG microsphere mixture to each well. 5. Add 1-5 µL of dH2O to each background well. 6. Add 1-5 µL of each PCR reaction to appropriate wells. 7. Cover the plate to prevent evaporation and denature at 96°C for 90 seconds. METHODS 1 & 2 ONLY. DO NOT PERFORM THIS STEP IF USING METHOD 3. * 8. Hybridize at 37-45°C for 30 minutes. * 9. Prepare Reporter Mix by diluting streptavidin-R-phycoerythrin to 8-10 µg/mL in 1X Tm Hybridization Buffer. 10. Add 70-75 µL to each well. Mix gently. 11. Incubate at 37-45°C for 15 minutes. 12. Analyze 70 µL at hybridization temperature on the Luminex analyzer according to the system manual. * These steps can be performed on a thermal cycler programmed as follows – Hold at 96°°C, 90 seconds Hold at 37°°C, FOREVER 3/15/2010 ADV-004 44 Hybridization to MagPlex-TAG Microspheres – No Wash Protocol for METHOD 3 Microspheres should be protected from prolonged exposure to light throughout this procedure. 1. Select the appropriate MagPlex-TAG microsphere sets and resuspend according to the instructions described in the Product Information Sheet provided with your microspheres. 2. Combine 2500 microspheres of each set per reaction. 3. Dilute/concentrate the MagPlex-TAG microsphere mixture to 125 of each microsphere set per µL in 1.1X Tm Hybridization Buffer by vortex and sonication for approximately 20 seconds. 4. Aliquot 20 µL of the MagPlex-TAG microsphere mixture to each well. 5. Add 1-5 µL of dH2O to each background well. 6. Add 1-5 µL of each PCR reaction to appropriate wells. 7. Prepare Reporter Mix by diluting streptavidin-R-phycoerythrin to 8-10 µg/mL in 1X Tm Hybridization Buffer. 8. Add 70-75 µL to each well. Mix gently. 9. Cover the plate to prevent evaporation and hybridize at 37-45°C for 25-45 minutes. * 10. Analyze 70 µL at hybridization temperature on the Luminex analyzer according to the system manual. * This step can be performed on a thermal cycler programmed as follows – Hold at 37°°C, FOREVER 3/15/2010 ADV-004 45 Hybridization to MagPlex-TAG Microspheres – Washed Protocol Microspheres should be protected from prolonged exposure to light throughout this procedure. 1. Select the appropriate MagPlex-TAG microsphere sets and resuspend according to the instructions described in the Product Information Sheet provided with your microspheres. 2. Combine 2500 microspheres of each set per reaction. 3. Dilute/concentrate the MagPlex-TAG microsphere mixture to 125 of each microsphere set per µL in 1.1X Tm Hybridization Buffer by vortex and sonication for approximately 20 seconds. 4. Aliquot 20 µL of the MagPlex-TAG microsphere mixture to each well. 5. Add 1-5 µL of dH2O to each background well. 6. Add 1-5 µL of each PCR reaction to the appropriate wells. 7. Cover the plate to prevent evaporation and denature at 96°C for 90 seconds. METHODS 1 & 2 ONLY. DO NOT PERFORM THIS STEP IF USING METHOD 3. * 8. Hybridize at 37-45°C for 30 minutes. * 9. Pellet the MagPlex-TAG microspheres by placing the plate on a magnetic separator and allow separation to occur for 30 to 60 seconds. Remove the supernatant. See Technical Note. 10. Resuspend the pelleted MagPlex TAG microspheres in 75 µL of 1X Tm Hybridization Buffer. 11. Repeat steps 11 and 12. This is a total of two washes. 12. Pellet the MagPlex-TAG microspheres by placing the plate on a magnetic separator and allow separation to occur for 30 to 60 seconds. Remove the supernatant. 13. Resuspend microspheres in 75 µL of 1X Tm Hybridization Buffer containing 2-8 µg/mL streptavidin-R-phycoerythrin. 14. Incubate at 37-45°C for 15 minutes. 15. Analyze 50 µL at hybridization temperature on the Luminex analyzer according to the system manual. * These steps can be performed on a thermal cycler programmed as follows – Hold at 96°°C, 90 seconds Hold at 37°°C, FOREVER 3/15/2010 ADV-004 46 Technical Note: Alternatively, wash steps can be performed by centrifugation or vacuum filtration. • Pellet the MagPlex-TAG microspheres by centrifugation at ≥ 2250 x g for 3 minutes and remove the supernatant. • Pre-wet a 1.2 µm Millipore filter plate with 1X Tm Hybridization Buffer and filter by vacuum manifold. Transfer the reactions to the pre-wetted filter plate and remove the supernatant by vacuum filtration. Wash twice with 100 µL 1X Tm Hybridization Buffer. Proceed with step 13. 3/15/2010 ADV-004 47