Butler, John M.

David, Victor A.

Menotti-Raymond, Marilyn

O\u27Brien, Stephen J.

English

NSU Works

Nova Southeastern University NSUWorks Biology Faculty Articles Department of Biological Sciences 9-2002 The MeowPlex: A New DNA Test Using Tetranucleotide STR Markers for the Domestic Cat John M. Butler National Institute of Standards and Technology Victor A. David National Cancer Institute at Frederick Stephen J. O'Brien National Cancer Institute at Frederick, sobrien1@nova.edu Marilyn Menotti-Raymond National Cancer Institute at Frederick Follow this and additional works at: https://nsuworks.nova.edu/cnso_bio_facarticles  Part of the Computer Sciences Commons, and the Genetics and Genomics Commons NSUWorks Citation Butler, John M.; Victor A. David; Stephen J. O'Brien; and Marilyn Menotti-Raymond. 2002. "The MeowPlex: A New DNA Test Using Tetranucleotide STR Markers for the Domestic Cat." Profiles in DNA 5, (2): 7-10. https://nsuworks.nova.edu/cnso_bio_facarticles/310 This Article is brought to you for free and open access by the Department of Biological Sciences at NSUWorks. It has been accepted for inclusion in Biology Faculty Articles by an authorized administrator of NSUWorks. For more information, please contact nsuworks@nova.edu. INTRODUCTIONWith over 65 million cats in the United States today (Pet Food Institute,Washington, D.C.), a large percentage of U.S. households have a cat and thus anabundance of cat hair. With the appropriate DNA tools, cat hairs could be used bya forensic laboratory to provide a link between the perpetrator of a crime and acrime scene. An assailant may unknowingly carry clinging cat hairs from a victim’scat away from the scene of a crime, or hair from the perpetrator’s cat may be leftat the scene (1). Either scenario may provide a crucial link and help solve animportant case. In the most famous case to date, DNA analysis of hair from a catnamed Snowball was used to link a murder suspect to a crime scene—a resultthat led to a criminal conviction (2).Evidence from domestic cat hairs may play an increasingly larger role in the futuredue to a new cat DNA test that has recently been developed through acollaboration between the National Cancer Institute’s (NCI) Laboratory of GenomicDiversity and the National Institute of Standards and Technology (NIST). Forseveral years, the Laboratory of Genomic Diversity has been working towards thedevelopment of this cat STR typing system. The goals of this project are theselection of polymorphic tetranucleotide STR loci with characteristics similar to the13 STR loci in use today for the FBI’s Combined DNA Index System (CODIS), thecreation of a robust multiplex STR assay for cats, and the development ofpopulation databases for various cat breeds. In the Snowball case, a set of 10 dinucleotide repeat markers was used withsingleplex PCR amplification (2,3). While a reliable match was made in thatparticular case, the high degree of stutter with dinucleotide repeats and theconsumption of precious DNA with singleplex amplifications are undesirable.Because many cat hairs yield very little DNA suitable for STR analysis, multiplexPCR will be critical for obtaining substantial results in future cases.SELECTION OF CAT STR MARKERSHundreds of STR markers have been mapped in the cat genome using techniquesdescribed previously (4). Initially, 22 potentially polymorphic tetranucleotide lociwere selected for further characterization and possible inclusion in a final forensicpanel. These STR markers were tested across approximately 200 cat DNAsamples representing 29 different breeds (i.e., 5–10 samples per breed; ref. 5).From these initial studies, 11 highly polymorphic tetranucleotide loci were selectedfor inclusion in a multiplex assay.Evidence fromdomestic cat hairsmay play anincreasingly largerrole in the future dueto a new cat DNAtest that has recentlybeen developed.7www.promega.com SEPTEMBER. 2002MEOWPLEXThe MeowPlex: A New DNA Test UsingTetranucleotide STR Markers for the Domestic CatBy John M. Butler1, Victor A. David2, Stephen J. O’Brien2, and Marilyn Menotti-Raymond2 1Biotechnology Division, National Institute of Standards and Technology, Gaithersburg,Maryland, and 2Laboratory of Genomic Diversity, National Cancer Institute-FrederickCancer Research and Development Center, Frederick, MarylandPromega 104022-Body Common  9/23/02  12:28 PM  Page 78SEPTEMBER. 2002 www.promega.comMEOWPLEXSelection criteria included Mendelianinheritance patterns, lack of linkagebetween loci (i.e., located on differentchromosomes or well-spaced on thesame chromosome), highheterozygosity across multiple breeds,no cross-species amplification, singletarget amplification, and absence ofnull alleles caused by sequencevariation in primer binding regions. A mutation rate of less than 1 in1,000 meioses was also a desirablecharacteristic, just as it is for humanSTR markers.The cat STR loci selected for theforensic panel are listed in Table 1along with their characteristics asdetermined in the initial study. TheF85 locus is the most polymorphicand has alleles that span a range ofmore than 100bp. It should be notedthat the names of the cat STRmarkers listed in Table 1 are notfinalized and thus may change as theyreceive “FCAxxx” designations.Cats have 18 pairs of autosomes andthe sex chromosomes X and Y. Catautosomes are designated A1, A2,A3, B1, B2, B3, B4, C1, C2, D1, D2,D3, D4, E1, E2, E3, F1, and F2,based on chromosome morphology,rather than the purely numericalformat from largest to smallest usedwith human chromosomes. Theforensic panel contains 11 STRs on 9different autosomes. The markers F53and B04 are on chromosome A1 whileF85 and G11 map to chromosomeB1. The cat STR loci that map to thesame chromosome are greater than50 centiMorgans apart based oncross-reference to tightly linkedmarkers that are mapped in theradiation hybrid and genetic linkagemaps of the cat (4,6). Thus, scientistsshould be able to combine the allelefrequencies for each individual locususing the product rule to obtain a highpower of genetic discrimination. THE MULTIPLEX STR ASSAYAll 11 cat STR markers supplied toNIST from NCI were incorporated intoa multiplex assay dubbed the“MeowPlex”. A gender identificationmarker was also included in themultiplex through the addition ofprimers specific for the SRY gene onthe cat Y chromosome (6). The PCRproducts from this 12plex multiplexamplification fall in the size range of100bp to 400bp and use three dyecolors. Primers were designed toleave approximately 20bp betweeneach locus in the same dye color toreduce potential overlap if new allelesare discovered in the future. Theallele size ranges and dye colors forthe MeowPlex assay are illustrated inFigure 1. Four loci are labeled in blue,five in green, and three in yellow.Primers amplify well using the samethermal cycling conditions used incommon commercial STR kits, namely28 cycles of PCR with an annealingTable 1. Cat STR Markers Used in the MeowPlex Assay. Results obtained from typing over 200cats representing 29 different breeds (5).Locus Dye Chromosomal Observed Heterozygosity Alleles Repeat Identifier Label Location (range across breeds) Observed MotifF53 Blue A1 0.53–0.93 9 Simple repeat[AAGA]C08 Blue B2 0.44–1.00 17 Compound repeat[ATAG][ATAC]B04 Blue A1 0.35–0.93 22 Compound repeat[AAGG][AAAG]G11 Blue B1 0.10–0.80 6 Simple repeat[ATCC]FCA441 Green D3 0.20–0.89 10 Simple repeat[ATAG]D09 Green B4 0.43–0.93 26 Compound repeat[ACAT][ATAG]F124 Green E1 0.51–0.93 20 Compound repeat[AGGA][AGAA]C12 Green F2 0.20–0.96 14 Complex repeat[AGAT][ACAT]C09 Yellow D4 0.00–0.94 15 Simple repeat[CTTT]F85 Yellow B1 0.66–0.98 33 Complex repeat[TTTC][TCTC]D06 Yellow C1 0.17–0.94 22 Simple repeat[TATC]SRY Green Y N/A +/– Gender IDThe use of familiaramplification conditionsand PCR set-up andperformance should ease the adoption of the MeowPlex assay byforensic DNA laboratoriesalready performing humanSTR typing.Promega 104022-Body Common  9/23/02  12:28 PM  Page 89www.promega.com SEPTEMBER. 2002MEOWPLEXtemperature of 59°C. The use offamiliar amplification conditions andPCR set-up and performance shouldease the adoption of the MeowPlexassay by forensic DNA laboratoriesalready performing human STR typing.The MeowPlex typically requires 1–5ng of extracted DNA for populationsamples (Figure 2). The assaysensitivity has not been testedextensively at subnanogram levels but should be similar to commercialhuman STR kits.The current dye labels used for theMeowPlex include 6 FAM (blue), VIC(green), and NED (yellow). Whenanalyzed with the ABI PRISM® 310Genetic Analyzer, these dyes arecompatible with filter set F (4-dye) ifthe GS500 ROX size standard isused, or with filter set G5 (5-dye) ifthe GS500 LIZ size standard is used.Matrix standards are alsocommercially available to create theappropriate color separation matriceson a variety of instruments. TheMeowPlex amplification products havebeen successfully analyzed using theABI PRISM® 377 DNA Sequencer andABI PRISM® 310, 3100, and 3700Genetic Analyzers at NIST and NCI.SPECIES SPECIFICITY WITHMEOWPLEX PRIMERSDNA samples from 29 differentspecies including humans have beentested with the MeowPlex in order todetermine species specificity (Menotti-Raymond et al., in preparation).Preliminary tests demonstrated thatthe MeowPlex is specific to individualsin the family Felidae (i.e., domesticcats and other felids such as pumasand ocelots). Monomorphic PCRproducts were observed in dog, wolf,and otter samples. No amplification is observed with human DNA usingthe MeowPlex primers. POPULATION STUDIESPopulation studies are underway atthe Laboratory of Genomic Diversitywhere DNA samples from over 1,200cats representing the 37 differentbreeds recognized by the two largestcat breed registries in the UnitedStates (the Cat Fanciers Associationand the International Cat Association)have been collected. An eight-generation pedigree has beenobtained for most of the cats in thedatabase so that levels of inbreedingand relatedness may be determined.Based on a preliminary population3783MA07_2ACat STR 12-plex("MeowPlex")100F53SRYD06F85C09C12F124D09FC0441C08 B04 G11200 300 400Figure 1. Schematic description of allele ranges and dye colors in the newly-developed cat STR12plex (MeowPlex).Figure 2. Meowplex PCR amplification of male and female cats. Four nanograms of DNAisolated from cat hair was amplified per reaction.On average, about 10ng of nuclear DNA can berecovered from the root of a single cat hair.3784CA07_2APromega 104022-Body Common  9/23/02  12:28 PM  Page 910SEPTEMBER. 2002 www.promega.comMEOWPLEXscreen of 29 breeds across 10 of the11 cat STRs (excluding G11), randommatch probabilities in the range of10–7 to 10–13 are observed. Thus, theMeowPlex has good potential forgenetic individualization of catsacross all breeds. This fact will beimportant as only ~3% of cats arepurebreds.RECOVERY OF DNA FROM CATHAIR ROOTSCat hair morphology and age appearto be associated with genotypingsuccess. Preliminary results fromexperiments conducted at NCIindicate that more than 50% of thetime STR results can be obtained from cat hairs containing roots. Onaverage, about 10ng of nuclear DNAcan be recovered from the root of asingle cat hair (5).CONCLUSIONA 12plex PCR multiplex has beendeveloped for genotyping 11 STRs and a gender identification marker indomestic cats. The MeowPlex worksunder similar conditions and hassimilar sensitivities to human STR kitsand is specific to cats and closerelatives. Ongoing population studiesshould greatly enhance use of thisnonhuman evidence. A commercial catDNA test, in a format similar tocurrent human STR analyses, could beavailable in the future to aid forensicDNA laboratories with analysis of thisvaluable evidence.ACKNOWLEDGMENTSThe authors gratefully acknowledgethe National Institute of Justice forfunding through interagencyagreements to the National CancerInstitute’s Laboratory of GenomicDiversity (development of domesticcat forensic typing system) and theNational Institute of Standards andTechnology’s Office of LawEnforcement Standards (developmentof multiplex PCR assays). WilliamMurphy supplied the SRY sequenceused to design the Y chromosome-specific PCR primers and mapped theSTRs in the cat genome using aradiation hybrid panel. Leslie Wachterand Amy Snyder provided excellenttechnical support for the project. Certain commercial equipment,instruments and materials areidentified in order to specifyexperimental procedures ascompletely as possible. In no casedoes such identification imply arecommendation or endorsement bythe National Institute of Standardsand Technology, nor does it imply thatany of the materials, instruments, orequipment identified are necessarilythe best available for the purpose.REFERENCES1. D’Andrea, F. et al. (1998) Preliminaryexperiments on the transfer of animal hairduring simulated criminal behavior. J. Forensic Sci. 43, 1257–1258.2. Menotti-Raymond, M. et al. (1997) Pet cathair implicates murder suspect. Nature 386,774.3. Menotti-Raymond, M. et al. (1997) Geneticindividualization of domestic cats using felineSTR loci for forensic applications. J. Forensic Sci. 42, 1039–1051.4. Menotti-Raymond, M. et al. (1999) A geneticlinkage map of microsatellites in thedomestic cat (Felis catus). Genomics 57,9–23.5. Menotti-Raymond, M. et al. Mapping,characterization and selection of a forensictyping panel of STR loci for geneticindividualization of domestic cat (Felis catus)samples. Manuscript in preparation.6. Murphy, W.J. et al. (2000). A radiation hybridmap of the cat genome: implications forcomparative mapping. Genome Res 10,691–702.ABI PRISM is a registered trademark of Applera Corporation. Promega 104022-Body Common  9/23/02  12:28 PM  Page 10

The MeowPlex:  A New DNA Test Using Tetranucleotide STR Markers for the Domestic Cat

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The MeowPlex: A New DNA Test Using Tetranucleotide STR Markers for the Domestic Cat

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