Humboldt-Universit(1)

1、International Society of Forensic GeneticsAugust 30, 2001John Butler Rich Schoske Pete ValloneHighly Multiplexed Assays for Measuring Polymorphisms on the Y-ChromosomeSTR MarkersDYS19DYS389I/IIDYS390DYS391DYS392DYS393DYS385YCAIIDYS437DYS438DYS439YThere is a growing interest in the Y-chromosome to ai

2、d forensic and paternity testing(50 presentations here at ISFG on Y markers)/genome/guide/Human Genome1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X YWe need:Population dataMultiplex assaysStandards/europe/European Y-STR Haplotype Reference Dat

3、abaseCreated by Sascha Willuweit and Lutz RoewerInstitute of Legal Medicine, Humboldt-Universitt Berlin, Germanyin cooperation with Michael Krawczak (Cardiff), Manfred Kayser (Leipzig) and Peter de Knijff (Leiden)This database has been accessed 14809 times since 01/01/2000. Last haplotype entry 3/26

4、/2001Current state of the database:45 European population samples5529 minimal haplotypes, 2196 of these are extended haplotypesn = 850 mt-DNA D-Loop sequences (data kindly provided by the Institute of Legal Medicine Magdeburg, Germany)n = 2196 extended European haplotypes logged in the databaseMore

5、markers = greater power of discriminationStandard Way to Type “Extended Haplotype”GDB primers2-3 multiplexes for minimal haplotypeDYS385 run separately sometimesYCAII run separately to obtain extended haplotypeDifferent PCR conditionsPrimers often require titrations (10 fold difference in amounts) i

6、n order to establish reasonable balance between Y STR PCR productsPublished primers were not designed to work together (originally used in single-plex PCRs)Commonly Used Y STR Multiplex Assay100 bp400 bp300 bp200 bpDYS19389II389I390Primer AmountsDyeY190.4 MTETY3890.12 MTETY3900.12 MFAMY3930.2 MHEXGu

7、smo et al. 1999 (Forensic Sci Int, vol. 106, pp. 163-172)5-plex393Higher level multiplexes are needed to improve the power of discrimination for Y chromosome DNA testsDye colorPCR product size rangeInefficient use of space across size range and dye colorAssay Development GoalsSingle tube amplificati

8、on of all loci in minimal (9-loci) or extended (11-loci) European Y STR haplotypesIncorporation of additional polymorphic markers recently publishedNo female amplification products (avoid X chromosome homology)Spacing between loci in same color to allow additional undiscovered alleles to be accommod

9、atedSimilar concentration of primers to produce balanced amplification productsSensitivity to 100 pg male DNA with 28 cycle PCRDiscussion with M. Prinz December 2000Development Strategy for Y STR MultiplexesCareful definition of allele rangesLiterature searches of over 200 papers to locate all known

10、 allelesEvaluation of diverse population samples to search for rare alleles (M. Hammer cell lines)Permits markers to be packed together more closely in a single dye colorAvoiding polymorphic nucleotides in primer binding sitesAlignment of multiple GenBank entriesPermits primers to be designed with l

11、ess risk of null alleles Incorporation of newly developed NIST multiplex assay design and testing toolsTools for MultiplexingPrimer design softwareVisual Basic programs to check potential primer dimer formationQuality control testing of primersButler et al. (2001) Forensic Sci. Int. 119: 87-96Rapid

12、multiplex testingButler et al. (2001) Fresenius J. Anal. Chem. 369: 200-205M96M45M9M89M42YCC Panel Sample Typed with Reliagene Y STR KitExtreme “off-ladder” allele5020015012517575100DNA Size (bp)A7.1H4DYS391DYS438DYS437DYS392DYS435DYS439DYS19DYS436NIST Y STR 10-plexPrimers were redesigned for most o

13、f these loci in order to keep the PCR products under 200 bp so that degraded DNA could be more successfully typed.Designed by Christian RuitbergPresented at Promega meeting October 2000436H4A7.143943543819392391437Dye blob436H4A7.143943543819392391437Dye blobDye blobMale 1Male 2Female426385II385I393

14、YCAII390392A7.1447391 19437389I389II 438448H4388439GS500 LIZ internal sizing standardNIST Y-STR 20plexDeveloped by Rich Schoske and John ButlerIncorporates all loci in the European Y STR “extended haplotype” plus a number of other useful markers275DYS391DYS426DYS389IDYS437DYS19DYS389IIDYS438DYS393YC

15、AIIDYS390DYS385A7.1H4DYS388DYS439DYS392DYS447DYS448Y STR 20plex utilizing 5 dye chemistryGenotyper resultEuropean “extended haplotype”6FAM (blue)VIC (green)NED (yellow)PET (red)100 bp200 bp300 bp400 bp75 bp438391389I43719389II6FAM (blue)393390385 I/II426YCAIIVIC (green)A7.1H4439392388NED (yellow)447

16、448PET (red)LIZ (orange)LIZ GS500-internal size standardDetails will be presented at the Promega meeting in October 2001Utilizes 5-dyesSame dyes as new ABI Identifiler kitSchematic of Loci in NIST Y STR 20-plexDesigned by Richard SchoskeLoci outlined in red have new primersEuropean “extended haploty

17、pe”100 bp200 bp300 bp400 bp75 bp391389I43719389II393390385 I/IIA7.1H443939291-119133-169184-196214-250257-289108-136191-227240-30499-119129-143204-224242-2727162533.271618277239167141417914AE616712438299-3346133881117153-171YCAII145-165 1727426 97A?447 448206-241273-30922172329VICPETNED6FAMSchematic

18、 of Loci in NIST Y STR 20-plexDesigned by Richard SchoskeAdvantages of Y STR 20plexSingle amplification of “extended haplotype”European loci: 19, 385 I/II, 389I, 389II, 390, 391, 392, 393, YCAII a/bAdditional loci: 426, A7.1, H4, 388, 437, 438, 439, 447, 448Sensitive to 100X female DNA10 loci with a

19、mplicons less than 200 bp in size to aid results with degraded DNARegions deleted in X homologMass spec primers designed for DYS391Allele with 9 TCTA repeats = 99 bpDYS391 Primer ImprovementsYX*Significant homology exists between X and Y We have designed primers to anneal to regions that only appear

20、 on the Y chromosome (target X deletion regions)Primers also work well to produce small PCR products that can be readily analyzed by mass specPower of DiscriminationLoci TestedDYS385 (1)19, 389I/II, 390, 393 (5)Reliagene Y-Plex 6 kit (6)Minimal haplotype (9)Extended haplotype (11)NIST Y STR 20plexUn

21、ique HaplotypesDiscrimination*186279838789Tested against 97 male DNA samples including YCC panel*results based on different combination from subsets from 20plex testing results/haplofreq.htm (Charles Brenner; chance of obtaining a non-unique haplotype)1:561:2101:4701:6001:9001:1100Equal amount of Fe

22、male DNA (no amplification)Male DNAResult obtained with ABI 3100 capillary array instrumentRed peaks are LTI 50-500 ROX internal size marker426393391A7.1H439019388437392439385I/II389II438389IMale-Specific Amplification with Y STR Megaplex (16plex with 4 dye chemistry)High-throughput Y STR Typing on

23、the ABI 3100 (16-capillary array)7680 data points in 24 hours using new Y STR 20plexCapability to double current size of European Y STR database in less than one week with one instrument10 ng5 ng2 ng1 ng0.5 ngSensitivity Study with new NIST Y STR 16-plex28 cycle PCR with sensitivity of 0.5 ngPlans w

24、ith our Y STR MegaplexCurrently being tested in 2 other labsM. Prinz - forensic samples with 4 dye 16plexM. Hammer - population studies with 5 dye 20plexConcordance studies with YCC sample panelAllele sequencing.developmental validationPresentation at ISFG and Promega meetingPublications with comple

25、te detailsCommercializing into kit form?Haplogroups produced by Y SNP genotypingUnderhill et al. (2000) Nature Genetics 26: 358-361M42M9M96M45M895154 Da5154 Da5467 Da5427 DaDepurination of primermass = 273 DaddCmass = 313 DaddGSample ASample BMS Data from Y SNP Marker M96MALDI-TOF MS data can be col

26、lected in 5-10 secondsVallone and Butler, Analysis of SNPs by MS, Encyclopedia of Mass Spectrometry, in pressMass Spec Y SNP Multiplex Assay(M9, M45, M89, and M96) M89297ddAM45297ddAM9313ddGM96313ddGPExExExExPPPCM55 Test Sample (H1)Luminex Bead Y SNP Multiplex Assay(9 Y SNPs plus amelogenin) Result

27、from Invitrogen beta-test kitMale-Specific Multiplex PCR at 17 Y SNP Loci17 plex PCRFemale sample (K562)Male sample (AM209)20 bp ladder200 bp300 bp400 bpCE Separation Time (min)Remaining primersY SNPs AmplifiedM2 A/G M60 +TM3 C/TM67 A/TM9 C/GM69 T/CM13 G/CM89 C/TM26 G/AM96 G/CM35 G/CM119 A/CM42 A/TM

28、122 T/CM45 G/AM145 G/AM55 T/CABCDEF426385II385I393YCAII390392A7.1447391 19437389I389II 438448H4388439SRM 2395 Candidates (Y STR 20plex Data)5 male samples selected to represent diverse combination of allelesFemale test sample yielded no amplificationGenotyper Results with Reliagene Y Plex 6 Kit on 5 male candidate samples for SRM 2395Short Tandem Repeat DNA Internet Database/biotech/strbaseSTRBaseY-Chromosome STR Information AvailableOver 200 publications on Y STRs & SNPs catalogedAllele informatio