Signet Y-SNP Identification System
Contents
1. ARLI C t l Better research by design Signet Y SNP Identification System v2 0 Y SNP Genotyping Core Kit The Signet Y SNP Identification System v2 0 comprises a core detection kit a broad screening kit and eleven individual genotyping kits The Core Kit contains the Control DNAs Exonuclease 5x Labeling Mix and Hybridization Solution The screening and typing kits contain the specific 2X PCR Mixes and Bead Mixes necessary to complete the analysis All kits are for 100 Reactions Name Cat Name Cat Y SNP Genotyping Core Kit 11710 100 Y SNP KLMN Genotyping Kit 11717 100 Y SNP Primary Screening Kit 11711 100 Y SNP 01 Genotyping Kit 11718 100 Y SNP AB Genotyping Kit 1712 100 Y SNP 02 Genotyping Kit 11719 100 Y SNP CD Genotyping Kit 1713 100 Y SNP PQ Genotyping Kit 11720 100 Y SNP E Genotyping Kit 1714 100 Y SNP R1 Genotyping Kit 11721 100 Y SNP FGHI Genotyping Kit 11715 100 Y SNP R2 Genotyping Kit 11722 100 Y SNP J Genotyping Kit 1716 100 Note for customers familiar with Marligen Signet products please pay attention to changes in the protocol An extensive Y SNP bibliography and Y SNP Haplotype Map are available from Marligen upon request from technical service Marligen com To Order 866 464 4900 301 874 4990 customer service marligen com Important Information The product you have received is authorized for laboratory research use only The product has not been qualified or found sa2. It is most important to keep the gate tight on the right hand side since this will eliminate microsphere doublets from the analysis 9 MRLIGEN BIOSCIENCES Better research by design 7 Clear the Single box under the Start button Make certain that the B1 row is highlighted then click Start The Luminex 100 will read the rest of the samples automatically 8 After data collection is complete select Save on the File menu The data will be saved in the newly created session folder NOTE If this step is omitted all data will be lost INTERPRETING RESULTS The use of MasterPlex GT software from MiraiBio for data analysis is strongly recommended MasterPlex GT conveniently automates the analysis of the output files produced by the Luminex Data Collector software The main output file of Luminex Data Collect is called Output csv and is located in the session folder that was named during session setup Qutput csv may also be imported into Microsoft Excel or similar spreadsheet programs for manual analysis The following parameters are recommended for data analysis with MasterPlex GT or for manual data analysis 1 Use the PCR reaction s without Taq polymerase as the negative controls This will ensure that the reagent background is subtracted from the data 2 The minimum number of events the threshold number of beads that must be recorded should have a value of at least 20 This will avoid chatter generated by small numb
3. 10 ul Significant cost savings can be achieved with minimal reduction in sensitivity 8 Remove primers with Exonuclease Add 1 ul Exonuclease to each amplification reaction and mix thoroughly Incubate at 37 C for 30 min Inactivate the enzyme by heating at 80 C for 20 min This step may be done conveniently in the thermal cycler lil Label the amplified DNA A fluorescent tag is incorporated into the amplicons using Taq polymerase in a cycled primer extension reaction Set these reactions up in a clean 96 well PCR plate The total reaction volume of each well or tube is 25 ul NOTE The Labeling Solution is light sensitive Keep shielded from light 1 Thaw the Y SNP Labeling Mix It is very important to vortex the Y SNP Labeling Mix thoroughly for 5 10 seconds prior to each use to dissolve any precipitate that may form during each freeze thaw cycle 2 Calculate the amount of Labeling Master Mix components that will be needed using the table below adding one or two extra reactions to your calculation to ensure that 6 To Order 866 464 4900 301 874 4990 customer service marligen com MRLIGED BIOSCIENCES Better research by design you have sufficient volume The same Y SNP Labeling Mix is used for all of the multiplexes Y SNP Labeling Master Mix Component Amount per X Number of Final Reaction Reactions Volume 1875W oo o Y SNP Labeling Mix Ph Taq DNA Polymerase 5 U l o2u O O 3 Prepare th
4. a software package such as MasterPlex GT from MiraiBio Overview The Marligen Y SNP Identification System is designed to determine the genotype of human male Y chromosomes Five simple steps comprise the assay 1 Amplify the polymorphic Y chromosome loci by multiplexed PCR Remove the PCR primers by incubating with Exonuclease Label the PCR products by extension from a fluorescently tagged oligonucleotide Hybridize the labeled DNA with allele specific oligonucleotides in a Bead Mix array of ee Om Read the hybridized microspheres in a Luminex 100 System The Y SNPs used to genotype the Y chromosome are grouped into 12 multiplexes for highly efficient determination of a Y chromosome genotype The multiplexes are summarized in Table 1 2 MRLIGEN To Order 866 464 4900 301 874 4990 customer service marligen com BIOSCIENCES Better research by design Table 1 Polymorphic loci in the Y SNP Identification System and their multiplex assignments Multiplex A R AB cb E FGHi J Multiplex Screening African Asian African Eurasian M Eastern description Multiplex 168C M168T M Eastern M89T M9G M304C M89C M96G Mg6C M304A M9 DYS391 M33 M35 M58 M75 M78 M81 M123 Multiplex Eurasian Asian Indo Indo description M9C M45G Native European European M175 American M207G M34 M343A M45A M207 3C A M88 M17 M18 M95 M56 M37 M101 M87 M65 M103 M124 M126 M119 M157 M153 P31 M173 M269 SRY465 SR
5. Y10831 P25 SRY 2627 The Multiplex A R SNPs define major groupings of Y haplogroups On samples of unknown origin it is recommended to first test the samples with the screening multiplex A R to determine the Y haplogroups of the samples The genotype can then be determined by subsequent testing with one of the Haplogroup specific multiplexes Additional Materials Required e _ Hot start Taq DNA Polymerase AmpliTaq Gold from Applied Biosystems or Platinum Taq from Invitrogen e Taq Polymerase e Water bath sonicator e Vortex mixer e Luminex 100 System with temperature controlled XY platform e 96 well PCR plate and plate sealer or PCR tubes with caps e _Temperature controlled block for 96 well plate set to 55 C Optional 3 To Order 866 464 4900 301 874 4990 customer service marligen com MRLIGED BIOSCIENCES Better research by design Storage of Components When you receive your Signet Y SNP Identification System you may store the entire kit at 20 C However prior to first use of the kit the components must be separated and stored under different conditions Core Kit Components Recommended Storage Voume 9947A and 9948 DNA 20 C Y SNP Labeling Mix 20 C protect from light Exonuclease 20 C Hybridization Solution Room Temperature Screening and Genotyping Kit Recommended Storage Components Y SNP 2X PCR Mixes 20 C PCR set up lab Y SNP Bead Mixes 4 C protect from light 200 pl Ge
6. conds and then sonicating for 1 minute 6 Add 37 ul of the Hybridization Bead Mix mixture to wells of the microtiter plate containing PCR product from the matching multiplex 7 Hybridize at 55 C for 30 minutes Protect samples from light V Read in Luminex 100 System Detailed instructions on hardware and software operations are provided in the Luminex 100 User s Manual The following is an abbreviated protocol specific for the Signet Y SNP Identification System For the bead map please see the file available on Marlgen s web site at www mariigen com 1 The Luminex 100 should have been prepared before the hybridization step A new session must be defined and the correct template loaded a From the Luminex Data Collector screen click Template on the toolbar b A dialog box will appear Select Signet Y chromosome DNA II and click OK 7 ARLIGEN N To Order 866 464 4900 301 874 4990 customer service marligen com BIOSCIENCES Better research by design 2 3 4 5 6 To Order 866 464 4900 301 874 4990 customer service marligen com NOTE If Signet Y chromosome DNA II does not appear on your list of templates contact Marligen by e mail at technical service marligen com The template and installation instructions will be sent to you via e mail Experienced users may use the bead map information from the Excel spreadsheet to key in their own template c A new dialog box will appear Enter a name to
7. e labeling master mix by pipetting the volumes of all components above into a sterile polypropylene tube Mix gently 4 Pipet 24 ll labeling master mix into the required number of sample and control wells or PCR tubes 5 Add 1 ul PCR product to each well containing labeling master mix 6 Cap or seal the PCR plate or tubes and place in a thermal cycler 7 Run the following Labeling Program Please note that the annealing temperature is 55 C rather than 58 C Initial Denaturation 94 C Per manufacturer s instructions 94 C 30 sec Amplification 40 cycles 55 C 30 sec 72 C 30 sec f 72 C 3 min Final Extension and Hold 5C Hold Samples may be stored overnight at 20 C after labeling IV Hybridize to Beads The Signet Y SNP Identification System contains Y SNP Bead Mixes that match the PCR mixes Make certain that a bead master mix is made for each multiplex or combination of multiplexes that were amplified Different multiplexes may be analyzed in the same plate 1 Warm up the Luminex 100 reader Follow the manufacturer s instructions for preparing and calibrating the instrument Using the Luminex Data Collector software set the temperature of the XY platform to 55 C Click on the Options icon followed by the Setup XY tab Make certain that the Heater Enabled box is 7 To Order 866 464 4900 301 874 4990 customer service marligen com MARLIGEN BIOSCIENCES Better research by design checked
8. elative DEBE Ga of E9 c GJ T Relative Intensity r Amel M122 M168 M175 M207 M304 M343 M45 M89 M96 M9 Figure 2 Plot of Relative Intensity for the alleles of a Test DNA sample with the A R multiplex The sample type is M9C M45G M175ins therefore this sample needs to be typed further with the KLMN multiplex 11 To Order 866 464 4900 301 874 4990 customer service marligen com MRLIGED BIOSCIENCES Better research by design 7 Figure 3 shows the genotyping result for the same test DNA sample tested with the KLMN multiplex The results show that the sample is M214C M231A TatC M178T i e its haplotype is N3a Relative Intensity of H11 100 f o uoo O QO tel ek Fk F Relative Intensity e8e 8 RF F Fz M178 M214 o z Figure 3 Plot of Relative Intensity for the alleles of a Test DNA sample with the KLMN multiplex The sample has been typed as M214C M231A TatC M178T and its haplotype is N3a Notes on some individual markers 1 Assays for some rare markers had been verified for the ancestral alleles only 2 P25 is a multi loci marker and will normally give a signal for both alleles Users should call alleles by the C to A signal ratio relying on the 9948 Control DNA which is P25A The haplotype of 9948 Control DNA is R1b3 The signal ratio C to A for P25 marker is approx 50 50 for A allele or 60 40 for C allele 3 The P37 allele may generate a signal
9. ers of stray microspheres 3 Use the relative intensity for allele calling To calculate the relative intensity of each allele the MFI of the allele is divided by the sum of the MFI of both alleles Rl a altele MFI A altete MF la altele MF lg altete 4 Figure 1 shows the raw MFI data from 85 individuals at the M2 locus collected on different days machines analysts and DNA inputs Although the MFI values vary considerably the values cluster tightly around lines that correspond to a relative intensity of 88 for the G allele or a relative intensity of 68 for the A allele 10 MRLIGED To Order 866 464 4900 301 874 4990 customer service marligen com BIOSCIENCES Better research by design Hybridization at Locus M2 RI G Allele 85 MFI M2 A Allele RI A Allele 68 MFI M2 G Allele Figure 1 Raw MFI signals at locus M2 Although MFI levels can vary relative intensity levels are nearly constant for each allele 5 The intensity threshold should be set to at least 20 Beads with a median fluorescent intensity MFI less than 20 will not be reported as an allele Samples with low intensity in all allelic forms such as female DNA will be reported as a null or no call 6 Figure 2 shows an example genotyping result for a test DNA that was tested with multiplex A R The results show that the sample is M9C M45G M1 75ins therefore this sample needs to be typed further with the KLMN multiplex R
10. fe and effective for any human or animal diagnostic or therapeutic application Uses other than the labeled intended use may be a violation of applicable law Precautions Warning This product contains hazardous reagents It is the end user s responsibility to consult the applicable MSDS s before using this product Disposal of waste organics acids bases and radioactive materials must comply with all appropriate federal state and local regulations If you have any questions concerning the hazards associated with this product please call Marligen Biosciences Inc at 301 874 4990 Trademarks Signet is a trademark of Marligen Biosciences Inc xMAP and Luminex are trademarks of Luminex Corporation MasterPlex is a trademark of MiraiBio Inc AmpliTaq Gold is a trademark of Applera Corporation Platinum is a trademark of Invitrogen Corporation Introduction to Signet Products Signet products and reagents are based on xMAP Suspension Array Technology DNA samples are amplified by PCR and labeled with a fluorescent tag The fluorescently labeled products are then hybridized to an array of allele specific oligonucleotides immobilized on latex beads Each bead is identified by a unique spectral address color After hybridization the Bead Mix is analyzed by the Luminex 100 system that records the spectral address of the bead and the amount of fluorescent tag bound to it Genotype reports can be generated by computer using
11. identify this session on the New Folder line and click OK d The main session screen will appear Click on the Setting button A dialog box will appear Enter the exact number of samples The operator name may also be entered The name of the session will appear in the Description box All of the other boxes contain information specified in the template e Events 150x the number of beads Total i e 1500 total for J 3300 total for A R etc Sample size 75 ul Min events 20 Flow rate fast Do not change any of these values Click OK to save the completed settings f The main session screen should now contain a row for every sample Descriptions of the samples may be entered by double clicking on the individual cell and typing in the information Place samples in the XY tray Fill the reservoir with sheath fluid and retract Run one wash cycle Check the Single box under the Start button Make certain that the reagent blank is highlighted and then click Start The instrument will read the Bead Only sample in well A1 Use the data from the reagent blank to set the gate a Make sure that the X axis of the histogram in the lower left corner is set to Doublet Discriminator b Right click on the histogram A pull down menu appears c Scroll to Gate and follow the arrow to the right d Click Create Dotted vertical red lines appear in the histogram frame e With the mouse drag each line to closely bracket the main peak
12. m Before beginning The use of gloves and aerosol resistant pipet tips is highly recommended Keep pre amplification and post amplification reagents in separate rooms Select the multiplexes to analyze On samples of unknown origin it is recommended to first test the samples with the screening multiplex A R to determine the Y haplogroups of the samples The genotype is then determined by subsequent testing with one of the Haplogroup specific multiplexes Different multiplexes may be run on the same PCR plate Thaw the Y SNP PCR Mixes It is very important to vortex the Y SNP PCR mixes thoroughly for 5 10 seconds prior to each use This dissolves any precipitate that may form during each freeze thaw cycle Check the Hybridization Solution for precipitate f a precipitate is present heat the Hybridization at 55 C and mix until dissolved Determine the number of reactions to be set up This should include a reagent blank no DNA and no Tag Polymerase a negative control no DNA added but with Taq and male DNA 9948 and female DNA 9947A controls Male DNA will yield a full profile while female DNA will only give signal on the amelogenin X bead in Multiplex 1 Add 1 or 2 reactions to the total number of reactions to compensate for losses during pipetting This will ensure that enough master mix is available for all samples in the experiment Amplify Y chromosome DNA Calculate the volumes of reagents needed for the master mix
13. neral Precautions Light Sensitivity The Labeling Mix and Bead Mixes in this product contain fluorescent dyes that are light sensitive It is safe to use these items in normal laboratory fluorescent lighting They should be covered and kept in a dark location when not in use Aluminum foil is useful for creating temporary light shields A drawer or darkened cabinet is also useful for short term storage during use Bead Settling Beads are 5 6 microns in diameter and are slightly more dense than water They will settle gradually and should be mixed just prior to any pipetting step Brief vortexing is sufficient for mixing suspensions that have settled for just a few minutes Upon storage for several hours small clumps of beads form that should be disrupted by sonication in a water bath The Luminex 100 has a mixing function built into it that briefly injects a few microliters of sheath fluid prior to analyzing a sample Therefore disruption is not necessary during the plate reading Critical Parameters e Use volumes precisely as indicated in the protocol Protect Labeling Mix and Bead Mixes from light Mix beads thoroughly to ensure a uniform suspension e Mix PCR and Labeling Reagent by vortexing prior to use 4 MRLIGEN To Order 866 464 4900 301 874 4990 customer service marligen com BIOSCIENCES Better research by design PROTOCOL 2 3 4 ll 1 2 To Order 866 464 4900 301 874 4990 customer service marligen co
14. the Set Point is 55 C and the aluminum 96 well plate holder is present in the XY platform NOTE The Luminex 100 XY platform is required for this procedure Make certain that the aluminum 96 well plate holder is installed and is pre heated to 55 C NOTE The Bead Mixes are light sensitive Protect Bead Mixes from the light whenever they are not in use 2 Add 35 ul Hybridization Solution to each well of the Costar 6509 96 well plate beginning at A1 and then continuing column by column until complete B1 H1 followed by A2 H2 etc 3 Transfer the labeled PCR product into wells containing Hybridization Solution Be certain to include the reagent blank controls and note their position A reagent blank will be used as part of the machine set up Add the remaining samples column by column A1 H1 followed by A2 H2 etc Place the plate into the pre heated block of the Luminex 100 XY Platform and incubate at 55 C while preparing Hybridization Bead Mix 4 Resuspend the Bead Mix by vortexing for 15 20 seconds Sonicate the suspension in a water bath sonicator for 2 minutes to create a single sphere suspension 5 Ina capped polypropylene tube prepare a Hybridization Bead Mix by adding 2 ul re suspended Y SNP Bead Mix to 35 ul of Hybridization Solution for each reaction Make sufficient Hybridization Bead Mix for 1 2 more reactions than the number you will be setting up Disperse the beads in the Hybridization Bead Mix by vortexing 15 20 se
15. using the table below Amount per X Number Reaction Of e Taq DNA Polymerase 5 U l Control or test DNA Component 1 10 E T 1 10 E T Deionized H20 0 9 ul To make 00 ul total volume Prepare the master mixes by pipetting the volumes of everything except sample from the above table into 1 5 or 2 ml polypropylene tubes Mix gently 5 MRLIGEN BIOSCIENCES Better research by design NOTE The procedure requires a hot start PCR protocol We recommend use of Platinum Taq from Invitrogen 3 Add 1 4 For the negative control pipette nuclease free water instead of DNA into the appropriate wells or tubes 10 ul of each DNA sample containing 1 8 ng into a well or tube 5 Pipette the appropriate volume of master mix into the wells or tubes 6 Cap or seal the PCR plate and place in a thermocycler 7 Run the following Amplification Program The duration of the initial 94 C step should be adjusted according to the instructions provided by the manufacturer of the Taq polymerase Initial Denaturation 94 C Per manufacturer s instructions 94 C 30 sec Amplification 30 cycles 58 C 20 sec 72 C 90 sec R 72 C 3 min Final Extension and Hold 5 C Hold Samples may be stored overnight at 4 C after the amplification step NOTE If high sensitivity is not required use 1 2 the recommended amount of Taq polymerase amount with the same reaction volume and or scale the reaction to a final volume of
16. with female DNA samples due to cross reactive sequences in the female genome However the results for P37 will be accurate when the sample comprises only male DNA 4 The assay for M17 will always develop strong signal for the de allele The signal for the ns allele is negligible for the ae type samples or approx 40 for the ns type samples Contact Us Marligen Biosciences Inc 301 874 4990 2502 Urbana Pike 301 874 4993 fax ljamsville MD 21754 www marligen com 20109 rev 081905 12 MRLIGEN To Order 866 464 4900 301 874 4990 customer service marligen com BIOSCIENCES Better research by design
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