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Text S3.
The minisequencing high throughput SNP genotyping strategy
Analysis of mtDNA SNPs always supposes a challenge for those laboratories carrying out tedious RFLP analysis of several dozens of coding region SNPs. In addition to the high cost and personal effort related to the genotyping of coding region SNPs, there is also an immanent risk for sample cross-over and contamination due to the number of PCR amplicons involved. In order to overcome this problem, the SNP genotyping carried out in the present study was based on a minisequencing technique which has been extremely useful in many clinical, forensic, and population genetic applications ADDIN EN.CITE Alvarez-Iglesias20082919291917lvarez-Iglesias, V.Barros, F.Carracedo, .Salas, A.Unidade de Xenetica, Instituto de Medicina Legal, Facultad de Medicina, Universidad de Santiago de Compostela, Galicia, Spain. vaneiml@usc.esMinisequencing mitochondrial DNA pathogenic mutationsBMC Med GenetBMC Med Genet269DNA Mutational Analysis/*methodsDNA PrimersDNA, Mitochondrial/*geneticsHumansLeigh Disease/geneticsMELAS Syndrome/geneticsOptic Atrophy, Hereditary, Leber/*genetics*Point MutationPolymerase Chain ReactionPolymorphism, Single NucleotideSpain200818402672http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=18402672 lvarez-Iglesias20072491249117lvarez-Iglesias, V.Jaime, J.C.Carracedo, .Salas, A.Coding region mitochondrial DNA SNPs: targeting East Asian and Native American haplogroupsForensic Sci Int: GenetForensic Sci Int: Genet44-5512007Quintns200499699617Quintns, B.lvarez-Iglesias, V.Salas, A.Phillips, C.Lareu, M. V.Carracedo, .Unidad de Genetica, Instituto de Medicina Legal, Universidad de Santiago de Compostela, San Francisco s/n, Santiago de Compostela, Galicia 15782, Spain.Typing of mitochondrial DNA coding region SNPs of forensic and anthropological interest using SNaPshot minisequencingForensic Sci IntForensic Sci Int251-71402-3DNA PrimersDNA, Mitochondrial/*analysisEuropeForensic Anthropology/*methodsForensic Medicine/*methodsHaplotypesHumanPolymerase Chain ReactionPolymorphism, Single NucleotideSupport, Non-U.S. Gov't*Variation (Genetics)2004Mar 1015036446http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=15036446(Quintns et al. 2004; lvarez-Iglesias et al. 2007; lvarez-Iglesias et al. 2008). The minisequencing reactions designed in the present study allow to genotype a total of 71 SNPs in three simple reactions. This method tries to avoid the hierarchical SNP genotyping approach which is the standard procedure in Y-chromosome SNP genotyping studies but also frequently exercised in mtDNA ones. Genotyping the whole set of SNPs in all the samples can also be useful to gain information about site-specific mutation rates and the phylogeny.
Several phylogenetic inconsistencies were observed in the present study (Table S2); some of these inconsistencies involved important diagnostic sites of the mtDNA phylogeny. For instance, G11719A is present in three H* samples (therefore all carry A2706G, C7028T, C14766T); two from Galicia (#C170 and #C203) and one from Catalonia (#AU87). Also interesting is the case of sample #AU74 from Catalonia. This sample belongs to HV (it lacks e.g. G11719A C14766T C16223T) and it carries A2706G and C7028T (it does not belong to haplogroup H). However, this sample carries G4580A which defines haplogroup V but it does not carries the necessary mutations C15904T and T16298C for this haplogroup. Sample #C25 belongs to H6a1 and carries the variant C15452T; note that the transversion C15452A defines haplogroup JT. To our knowledge there is only one record for C15452T, but since it occurs within haplogroup J ADDIN EN.CITE Uusimaa20042278227817Uusimaa, J.Finnil, S.Remes, A. M.Rantala, H.Vainionp, L.Hassinen, I. E.Majamaa, K.Department of Departments of Pediatrics, University of Oulu, Oulu, Finland.Molecular epidemiology of childhood mitochondrial encephalomyopathies in a Finnish population: sequence analysis of entire mtDNA of 17 children reveals heteroplasmic mutations in tRNAArg, tRNAGlu, and tRNALeu(UUR) genesPediatricsPediatrics443-501142Base SequenceChildDNA, Mitochondrial/*geneticsEpidemiology, MolecularFemaleHumansMaleMental Retardation/geneticsMitochondrial Encephalomyopathies/*geneticsMolecular Sequence DataRNA, Transfer/*geneticsResearch Support, Non-U.S. Gov'tSequence Analysis, DNASpeech Disorders/genetics2004Aug15286228http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=15286228 (Uusimaa et al. 2004), it could just represent a documentation error from the necessary transversion at this position.
References
ADDIN EN.REFLIST lvarez-Iglesias V, Jaime JC, Carracedo , Salas A (2007) Coding region mitochondrial DNA SNPs: targeting East Asian and Native American haplogroups. Forensic Sci Int: Genet 1: 44-55.
lvarez-Iglesias V, Barros F, Carracedo , Salas A (2008) Minisequencing mitochondrial DNA pathogenic mutations. BMC Med Genet 9: 26.
Quintns B, lvarez-Iglesias V, Salas A, Phillips C, Lareu MV et al. (2004) Typing of mitochondrial DNA coding region SNPs of forensic and anthropological interest using SNaPshot minisequencing. Forensic Sci Int 140(2-3): 251-257.
Uusimaa J, Finnil S, Remes AM, Rantala H, Vainionp L et al. (2004) Molecular epidemiology of childhood mitochondrial encephalomyopathies in a Finnish population: sequence analysis of entire mtDNA of 17 children reveals heteroplasmic mutations in tRNAArg, tRNAGlu, and tRNALeu(UUR) genes. Pediatrics 114(2): 443-450.
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