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A SNaPshot multiplex typing strategy for mtDNA identification of mouse inbred strains
Available online at www.sciencedirect.com
Forensic Science International: Genetics Supplement Series 1 (2008) 596–597 www.elsevier.com/locate/FSIGSS
Research article
A SNaPshot multiplex typing strategy for mtDNA identification of mouse inbred strains Ana Goios a,b,*, Leonor Gusma˜o a, Ana Mafalda Rocha a, Luı´sa Pereira a,c, Molly Bogue d, Anto´nio Amorim a,b a
IPATIMUP, Instituto de Patologia e Imunologia Molecular da Universidade do Porto, Porto, Portugal b Faculdade de Cieˆncias da Universidade do Porto, Porto, Portugal c Medical Faculty, University of Porto, Porto, Portugal d The Jackson Laboratory, Bar Harbor, ME, USA Received 13 August 2007; received in revised form 11 October 2007; accepted 12 October 2007
Abstract Mouse inbred strains have been continuously used for research in numerous fields in laboratories throughout the world. Rapid genotyping methods that allow the distinction of the different strains are important for both the distinction of materials such as tissue and cell collections and to identify the origin of new strains. Taking advantage of the homogeneity of mitochondrial DNA (mtDNA) sequences inside each inbred strain, and of the few mtDNA polymorphisms that differentiate inbred strains, we are reporting a strategy for identification of mtDNA haplotypes of a selected group of priority strains. We present a preliminary version of a SNaPshot multiplex typing strategy that, with only a pair of reactions, allows the distinction between common inbred and wild-derived mice strains, and provides the identification of 10 different common inbred and six wild-derived mice mtDNA haplotypes. We believe that this SNaPshot typing strategy will be of use for researchers that work regularly with mice strains and/or mouse tissues or cell lines. Moreover, it may also prove valuable in forensic identification of materials collected in laboratory accidents as well as in scientific fraud. # 2008 Elsevier Ireland Ltd. All rights reserved. Keywords: mtDNA; SNP; Mouse
1. Introduction More than 450 inbred mouse strains with different characteristics are used throughout the world in a wide variety of research fields. In many cases, two or more different strains coexist in the same laboratory facility. We aimed to develop a quick genotyping strategy that allows the distinction of different strains for use in cases of material mix ups or identification of the mother strain. Such strategy would also be useful in detecting laboratory fraud and refining case/control studies. Recently there were published mitochondrial DNA (mtDNA) sequences of various strains [1] that allowed identifying the single nucleotide polymorphisms (SNPs) that distinguish them. All individuals of a given inbred strain are
* Corresponding author at: IPATIMUP, R. Dr. Roberto Frias, s/n, 4200-465 Porto, Portugal. Tel.: +351 225570700; fax: +351 225570799. E-mail address: [email protected] (A. Goios). 1875-1768/$ – see front matter # 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.fsigss.2007.10.204
expected to present the same mtDNA sequence, making of mtDNA SNPs a good choice of markers. 2. Materials and methods We focused on 15 strains selected by the Mouse Phenome Project [1] as priority strains. We added to the alignments eight additional strains for which the mtDNA has been sequenced. Polymorphisms (see Supplementary Table 3) were identified by aligning the different sequences, and confronting with phylogenetic networks and trees from Goios et al. [2]. All primers (see Supplementary Table 1) and probes (see Supplementary Table 2) were primarily designed for the reference C57BL/6J sequence, the most widely used of all strains, avoiding, when possible, polymorphic regions, and following guidelines in Sanchez et al. [3]. A nine-plex PCR amplification was carried using QIAGEN Multiplex PCR Kit (Qiagen, Hilden, Germany), according to manufacturer’s
A. Goios et al. / Forensic Science International: Genetics Supplement Series 1 (2008) 596–597
instructions, and product was purified with ExoSAP-IT (Amersham Biosciences, Uppsala, Sweden). Two SNaPshot reactions (SBE1 and SBE2) were then performed using the SNaPshot Minisequencing Reaction Kit (Applied Biosystems, Foster City, CA), followed by purification with SAP (Amersham Biosciences). Final products were run in an ABI PRISM 3130 Genetic Analyzer with GeneScan-120 LIZ size standard, and results were analyzed with GeneMapper 4.0 (Applied Biosystems). 3. Results and discussion This SNaPshot multiplex strategy was tested in 16 priority inbred mouse strains (first 16 in see Supplementary Table 3) allowing the distinction of their mtDNA haplotypes in a single multiplex amplification and two SNaPshot reactions. Example of the profiles can be seen in see Supplementary Fig. 1. The multiplex/SNaPshot design was made in order to distinguish seven additional strains not included in this report. All probes yielded the expected result for the reference strain, C57BL/6J. A weak or absent amplification of some markers in wild-derived strains could be explained by a polymorphism in the probe annealing site. The presence of these ‘‘null alleles’’ is in itself part of the profile that should be obtained for these strains. Mobility of the shortest fragments (up to 35 bp long) is not proportional to fragment size, but it is likely influenced by factors such as GC content, molecular weight or secondary structures. This is worth investigating when designing a multiplex, so that the distribution of the markers is as even as possible.
597
4. Conclusions This SNaPshot multiplex currently allows the identification of 16 of the most widely used inbred mouse strains, and it can be easily reproduced for identification where needed. Nevertheless, we will continue testing it in other strains. Acknowledgments AG has a PhD grant (SFRH/BD/16518/2004) from Fundac¸a˜o para a Cieˆncia e a Tecnologia. This work was partially supported by ‘‘Programa Operacional Cieˆncia e Inovac¸a˜o 2010’’ (POCI 2010). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.fsigss.2007.10.204. Conflict of interest None. References [1] H. Pearson, Mouse sequencing plan aims to boost models, Nature 432 (2004) 5. [2] A. Goios, L. Pereira, M. Bogue, V. Macaulay, A. Amorim, mtDNA phylogeny and evolution of laboratory mouse strains, Genome Res. 17 (2007) 293–298. [3] J. Sanchez, C. Børsting, N. Morling, Typing of Y chromosome SNPs with multiplex PCR methods, in: A. Carracedo (Ed.), Methods in Molecular Biology, Forensic DNA Typing Protocols, vol. 297, Humana Press Inc., Totowa NJ, 2004, pp. 209–228.
Forensic Science International: Genetics Supplement Series 1 (2008) 596–597 www.elsevier.com/locate/FSIGSS
Research article
A SNaPshot multiplex typing strategy for mtDNA identification of mouse inbred strains Ana Goios a,b,*, Leonor Gusma˜o a, Ana Mafalda Rocha a, Luı´sa Pereira a,c, Molly Bogue d, Anto´nio Amorim a,b a
IPATIMUP, Instituto de Patologia e Imunologia Molecular da Universidade do Porto, Porto, Portugal b Faculdade de Cieˆncias da Universidade do Porto, Porto, Portugal c Medical Faculty, University of Porto, Porto, Portugal d The Jackson Laboratory, Bar Harbor, ME, USA Received 13 August 2007; received in revised form 11 October 2007; accepted 12 October 2007
Abstract Mouse inbred strains have been continuously used for research in numerous fields in laboratories throughout the world. Rapid genotyping methods that allow the distinction of the different strains are important for both the distinction of materials such as tissue and cell collections and to identify the origin of new strains. Taking advantage of the homogeneity of mitochondrial DNA (mtDNA) sequences inside each inbred strain, and of the few mtDNA polymorphisms that differentiate inbred strains, we are reporting a strategy for identification of mtDNA haplotypes of a selected group of priority strains. We present a preliminary version of a SNaPshot multiplex typing strategy that, with only a pair of reactions, allows the distinction between common inbred and wild-derived mice strains, and provides the identification of 10 different common inbred and six wild-derived mice mtDNA haplotypes. We believe that this SNaPshot typing strategy will be of use for researchers that work regularly with mice strains and/or mouse tissues or cell lines. Moreover, it may also prove valuable in forensic identification of materials collected in laboratory accidents as well as in scientific fraud. # 2008 Elsevier Ireland Ltd. All rights reserved. Keywords: mtDNA; SNP; Mouse
1. Introduction More than 450 inbred mouse strains with different characteristics are used throughout the world in a wide variety of research fields. In many cases, two or more different strains coexist in the same laboratory facility. We aimed to develop a quick genotyping strategy that allows the distinction of different strains for use in cases of material mix ups or identification of the mother strain. Such strategy would also be useful in detecting laboratory fraud and refining case/control studies. Recently there were published mitochondrial DNA (mtDNA) sequences of various strains [1] that allowed identifying the single nucleotide polymorphisms (SNPs) that distinguish them. All individuals of a given inbred strain are
* Corresponding author at: IPATIMUP, R. Dr. Roberto Frias, s/n, 4200-465 Porto, Portugal. Tel.: +351 225570700; fax: +351 225570799. E-mail address: [email protected] (A. Goios). 1875-1768/$ – see front matter # 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.fsigss.2007.10.204
expected to present the same mtDNA sequence, making of mtDNA SNPs a good choice of markers. 2. Materials and methods We focused on 15 strains selected by the Mouse Phenome Project [1] as priority strains. We added to the alignments eight additional strains for which the mtDNA has been sequenced. Polymorphisms (see Supplementary Table 3) were identified by aligning the different sequences, and confronting with phylogenetic networks and trees from Goios et al. [2]. All primers (see Supplementary Table 1) and probes (see Supplementary Table 2) were primarily designed for the reference C57BL/6J sequence, the most widely used of all strains, avoiding, when possible, polymorphic regions, and following guidelines in Sanchez et al. [3]. A nine-plex PCR amplification was carried using QIAGEN Multiplex PCR Kit (Qiagen, Hilden, Germany), according to manufacturer’s
A. Goios et al. / Forensic Science International: Genetics Supplement Series 1 (2008) 596–597
instructions, and product was purified with ExoSAP-IT (Amersham Biosciences, Uppsala, Sweden). Two SNaPshot reactions (SBE1 and SBE2) were then performed using the SNaPshot Minisequencing Reaction Kit (Applied Biosystems, Foster City, CA), followed by purification with SAP (Amersham Biosciences). Final products were run in an ABI PRISM 3130 Genetic Analyzer with GeneScan-120 LIZ size standard, and results were analyzed with GeneMapper 4.0 (Applied Biosystems). 3. Results and discussion This SNaPshot multiplex strategy was tested in 16 priority inbred mouse strains (first 16 in see Supplementary Table 3) allowing the distinction of their mtDNA haplotypes in a single multiplex amplification and two SNaPshot reactions. Example of the profiles can be seen in see Supplementary Fig. 1. The multiplex/SNaPshot design was made in order to distinguish seven additional strains not included in this report. All probes yielded the expected result for the reference strain, C57BL/6J. A weak or absent amplification of some markers in wild-derived strains could be explained by a polymorphism in the probe annealing site. The presence of these ‘‘null alleles’’ is in itself part of the profile that should be obtained for these strains. Mobility of the shortest fragments (up to 35 bp long) is not proportional to fragment size, but it is likely influenced by factors such as GC content, molecular weight or secondary structures. This is worth investigating when designing a multiplex, so that the distribution of the markers is as even as possible.
597
4. Conclusions This SNaPshot multiplex currently allows the identification of 16 of the most widely used inbred mouse strains, and it can be easily reproduced for identification where needed. Nevertheless, we will continue testing it in other strains. Acknowledgments AG has a PhD grant (SFRH/BD/16518/2004) from Fundac¸a˜o para a Cieˆncia e a Tecnologia. This work was partially supported by ‘‘Programa Operacional Cieˆncia e Inovac¸a˜o 2010’’ (POCI 2010). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.fsigss.2007.10.204. Conflict of interest None. References [1] H. Pearson, Mouse sequencing plan aims to boost models, Nature 432 (2004) 5. [2] A. Goios, L. Pereira, M. Bogue, V. Macaulay, A. Amorim, mtDNA phylogeny and evolution of laboratory mouse strains, Genome Res. 17 (2007) 293–298. [3] J. Sanchez, C. Børsting, N. Morling, Typing of Y chromosome SNPs with multiplex PCR methods, in: A. Carracedo (Ed.), Methods in Molecular Biology, Forensic DNA Typing Protocols, vol. 297, Humana Press Inc., Totowa NJ, 2004, pp. 209–228.