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Sequencing of mitochondrial DNA and the problem of human specificity
Forensic Science International: Genetics Supplement Series 2 (2009) 95–96
Contents lists available at ScienceDirect
Forensic Science International: Genetics Supplement Series journal homepage: www.elsevier.com/locate/FSIGSS
Research article
Sequencing of mitochondrial DNA and the problem of human specificity Rebecca Renneberg a,c,*, Anke Heinreich b, Ben Krause-Kyora b,c, Melanie Ro¨pke b,c, Jens Tetens d, Eva Simeoni b, Thorsten Schwark b, Nicole von Wurmb-Schwark b a
Institute of Experimental Medicine, CAU Kiel & University Hospital Schleswig-Holstein, c/o Arnold-Heller-Str. 3, House 28, D-24105 Kiel, Germany Institute of Legal Medicine, CAU Kiel & University Hospital Schleswig-Holstein, Arnold-Heller-Str. 3, House 28, D-24105 Kiel, Germany c Graduate School Human Development in Landscapes, Christian-Albrechts-University Kiel, Neufeldstraße 10, D-24118 Kiel, Germany d Institute of Animal Breeding and Husbandry, Christian-Albrechts-University Kiel, Hermann-Rodewald-Straße 6, D-24098 Kiel, Germany b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 21 August 2009 Accepted 26 August 2009
When analysing trace materials and degraded DNA the issue of human specificity is highly important. Especially when it comes down to the analysis of mitochondrial DNA which is extremely susceptible to contamination authenticity is the main question. Therefore in the presented study mitochondrial primers were tested on their human specificity. In all cases it was possible to amplify DNA of animals with human mt-primers. These unintentional amplifications could only be decreased by choosing austere PCR parameters. The study implies the importance of comprehensive evaluation of primers, chemicals and PCR parameters. ß 2009 Elsevier Ireland Ltd. All rights reserved.
Keywords: Mitochondrial DNA Human specificity
1. Case report In a forest near Flensburg two walkers found some sort of tissue wrapped in a blanket, next to several pieces of kitchen paper with apparent blood stains on it. They assumed that these were parts of a human placenta. The police immediately send some officers with track hounds – specific to human tissues – to the finding place. The dogs reacted to the tissue but did not find anything else. The tissues as well as other trace materials were brought to the Institute of Legal Medicine. After a positive blood pre-test, the Hexagon OBTI test showed negative results for the tissue and the bloodstains. Because false negative results with the OBTI test were reported before [1], DNA extraction was carried out and a human specific screening PCR [2], a duplex PCR [3] as well as two commercially available PCRs were performed. The screening PCR showed positive results for mitochondrial DNA, and the duplex PCR showed a positive signal for both the mitochondrial as well as the chromosomal fragment of the beta globin gene. In the commercial PCRs no consensus and reproducible signals could be detected. The ambivalent results were ascribed to a low and highly degraded DNA content. Since the police were certain of the human origin of the materials, because of the dog’s reaction, mitochondrial DNA sequencing was done. The sequence quality was poor and only a non-valuable sequence was obtained.
* Corresponding author at: Institute of Experimental Medicine, CAU Kiel & University Hospital Schleswig-Holstein, c/o Arnold-Heller-Str. 3, House 28, D24105 Kiel, Germany. Tel.: +49 4315973571; fax: +49 431 5973612. E-mail address: [email protected] (R. Renneberg). 1875-1768/$ – see front matter ß 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.fsigss.2009.08.155
After rethinking the results, as well as the fact that animal hair was found on the blanket, a DNA sample was sent to the Institute of Animal Breeding and Husbandry for additional analysis. Just a few days later the case was closed: the tissue belonged to the placenta of a cow. Due to the fact that the track hounds as well as the chosen PCRs were supposed to be human specific the process of analysis was unnecessarily prolonged. In consequence the used primers, especially mitochondrial primers, were retested for human specificity. 2. Materials and methods DNA from tissue as well as buccal swabs from 13 different species was extracted by an automated extraction (EZ1biorobot, Qiagen). Mitochondrial DNA was amplified by two different mtprimer pairs [4,5]. Cycling parameters were used with adjusted parameters for highly degraded material: 94 8C 5 min followed by 40 cycles of 94 8C 1 min, 57 8C 1 min, 72 8C 1 min for primer set I (expected amplicon length of 279 and 435 bp [4]) and for the primer set II (length of 179 and 434 bp [5]) 95 8C 10 min followed by 40 cycles of 94 8C 1 min, 57 8C 1 min and 72 8C 1 min respectively. Electrophoresis of the PCR products was performed on 2.5% agarose gels and a 6% PAGE. Sequencing was carried out on an ABI Prism310. Next to the sequence process the primer pairs were labelled with fluorescent dye (6-Fam) and applied on the samples, to get the exact fragment-length of PCR products by electrophoresis on the ABI Prism310.
96
R. Renneberg et al. / Forensic Science International: Genetics Supplement Series 2 (2009) 95–96
Table 1 Amplicons detected on an ABI310 after amplification of two mitochondrial DNA fragments with different temperatures and 40 cycles. Species
Amplicons (bp) with 57 8C AnnealT
Amplicons (bp) with 62 8C AnnealT
Homo sapiens Canis familiaris Python sp. Macaca sp. Cervus sp. Ovis aries Bos taurus Sus scrofa Anas sp. Pteropus sp. Pelicanus sp. Castor fiber Felis silvestris Gallus sp.
279, 435 136, 228, 246, 279, 483 156, 160, 166, 177, 279 90, 100, 161, 278, 434, 461 95, 112, 143, 278, 299, 336, 449, 486 278, 300, 482 196, 226, 279, 336, 494 265, 459 158, 266, 279, 306, 532 126, 278 278 438 278 129
279, 435 / / 279 / / / / / / / / / /
/ = no signals detected.
To carry out the optimal conditions for a highly specific PCR a temperature gradient and different numbers of cycles were performed. Additionally, a real-time PCR (which was tested on human specificity before) was carried out on the different samples to exclude the possibility of dealing with contaminant human DNA. In all PCRs a positive control as well as no template controls by a 1:5 ratio were carried along. 3. Results and discussion All samples showed positive results on the agarose gel and PAGE when using the annealing temperatures given in the literature and performing 40 cycles. In most of the samples next to the specific product several other unspecific fragments were obtained. None of the samples could be sequenced in a sufficient manner. The amplicons of the fluorescent labelled primers showed that the several fragments were between 90 and 532 bp long (Table 1). Fragment sizes show that the amplicons might not be derived by mitochondrial DNA; moreover searching with the BLAST tool (NCBI) revealed no complete match of the primers but several possibilities for generating false-positive results due to non-severe PCR parameters and low efficiency of the amplification. Table 1 shows the success rate in enlarging the specificity of the primers by increasing the temperature by 5 8C. Only the phylogenetic closest relative in the sample pool showed positive results due to the fact that the sequence of the primer binding site is very similar. Searching the sequence of Macaca 11 bp of the upper primer and 12 bp of the lower primer matched and matching sites were located at the 50 end and therefore allowed an amplification of the fragment. By reducing the cycle number, the amount of amplified product was reduced in species which are distantly related phylogenetically (birds and reptiles; data not shown). Here the amplification was caused by contaminations. 40 cycles are enough to amplify a single cell over the detection limit of agarose gels and therefore slightest traces of mitochondrial DNA contaminations become visible and might be interpreted as a positive result. These contaminations can originate from PCR tubes and may therefore occur in every PCR but will only become visible if the amount of DNA was high or adequate cycles were carried out to up-amplify the low amount of DNA. In our case real-time PCR proved that no contaminating human DNA was present in the DNA extracts of animal tissues used for these experiments. Therefore the amplified fragments originated from the samples. As a matter of fact false-positive results when analysing unidentified trace materials might occur.
4. Conclusion The analysis of mitochondrial DNA is highly susceptible for contaminations and should therefore be carried out with special cautiousness. But not only environmental conditions as well as contamination prevention are of importance, PCR itself must be ideally optimized to provide specific and highly reliable results. Even when primers are chosen carefully and are human specific, they are applicable on different species if using wrong PCR parameters. The main focus during the design of a new PCR or/and during the adoption of a new system in the lab should be on the specific annealing temperature which might differ from the one quoted by the manufacturer. Moreover, if sequencing results show highly unspecific fragments it is not only a sign of highly degraded DNA material but might also indicate an insufficient amplification. When dealing with material which cannot be determined as human material by morphological, histological or chemical/ physical analysis in general a genetic species determination should be applied first, e.g. [6,7]. Role of funding University research funding. Conflict of interest None. References [1] E. Johnston, C.E. Ames, K.E. Dagnall, J. Foster, B.E. Daniel, Comparison of presumptive blood test kits including hexagon OBTI, J. Forensic Sci. 53 (3) (2008) 687–689. [2] N. von Wurmb-Schwark, A. Preusse-Prange, A. Heinrich, E. Simeoni, T. Bosch, T. Schwark, A new multiplex-PCR comprising autosomal and Y-specific STRs and mitochondrial DNA to analyze highly degraded material, Forensic Sci. Int. Genet. 3 (2) (2009) 96–103. [3] N. von Wurmb-Schwark, T. Schwark, M. Harbeck, M. Oehmichen, A simple duplexPCR to evaluate the DNA quality of anthropological and forensic samples prior short tandem repeat typing, Legal Med. 6 (2004) 80–88. [4] H. Pfeiffer, P. Forster, C. Ortmann, B. Brinkmann, The results of an mtDNA study of 1200 inhabitants of a German village in comparison to other Caucasian databases and its relevance for forensic casework, Int. J. Legal Med. 114 (2001) 169–172. [5] L. Fehren- Schmitz, M. Reindel, E. Tomasto Cagigao, S. Hummel, B. Herrmann, PreColumbian Population Dynamics in Coastal Southern Peru: A Diachronic Investigation of mtDNA Patterns in the Palpa region by Ancient DNA Analysis, Am J Phys Anthropol (2009), doi:10.1002/ajpa.21135. [6] W. Parson, K. Pegoraro, H. Niedersta¨tter, M. Fo¨ger, M. Steinlechner, Species identification by means of the cytochrome b gene, Int. J. Legal Med. 114 (1–2) (2000) 23–28. [7] H.M. Hsieh, H.L. Chiang, L.C. Tsai, S.Y. Lai, N.E. Huang, A. Linacre, J.C. Lee, Cytochrome b gene for species identification of the conservation animals, Forensic Sci. Int. 122 (1) (2001) 7–18.
Contents lists available at ScienceDirect
Forensic Science International: Genetics Supplement Series journal homepage: www.elsevier.com/locate/FSIGSS
Research article
Sequencing of mitochondrial DNA and the problem of human specificity Rebecca Renneberg a,c,*, Anke Heinreich b, Ben Krause-Kyora b,c, Melanie Ro¨pke b,c, Jens Tetens d, Eva Simeoni b, Thorsten Schwark b, Nicole von Wurmb-Schwark b a
Institute of Experimental Medicine, CAU Kiel & University Hospital Schleswig-Holstein, c/o Arnold-Heller-Str. 3, House 28, D-24105 Kiel, Germany Institute of Legal Medicine, CAU Kiel & University Hospital Schleswig-Holstein, Arnold-Heller-Str. 3, House 28, D-24105 Kiel, Germany c Graduate School Human Development in Landscapes, Christian-Albrechts-University Kiel, Neufeldstraße 10, D-24118 Kiel, Germany d Institute of Animal Breeding and Husbandry, Christian-Albrechts-University Kiel, Hermann-Rodewald-Straße 6, D-24098 Kiel, Germany b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 21 August 2009 Accepted 26 August 2009
When analysing trace materials and degraded DNA the issue of human specificity is highly important. Especially when it comes down to the analysis of mitochondrial DNA which is extremely susceptible to contamination authenticity is the main question. Therefore in the presented study mitochondrial primers were tested on their human specificity. In all cases it was possible to amplify DNA of animals with human mt-primers. These unintentional amplifications could only be decreased by choosing austere PCR parameters. The study implies the importance of comprehensive evaluation of primers, chemicals and PCR parameters. ß 2009 Elsevier Ireland Ltd. All rights reserved.
Keywords: Mitochondrial DNA Human specificity
1. Case report In a forest near Flensburg two walkers found some sort of tissue wrapped in a blanket, next to several pieces of kitchen paper with apparent blood stains on it. They assumed that these were parts of a human placenta. The police immediately send some officers with track hounds – specific to human tissues – to the finding place. The dogs reacted to the tissue but did not find anything else. The tissues as well as other trace materials were brought to the Institute of Legal Medicine. After a positive blood pre-test, the Hexagon OBTI test showed negative results for the tissue and the bloodstains. Because false negative results with the OBTI test were reported before [1], DNA extraction was carried out and a human specific screening PCR [2], a duplex PCR [3] as well as two commercially available PCRs were performed. The screening PCR showed positive results for mitochondrial DNA, and the duplex PCR showed a positive signal for both the mitochondrial as well as the chromosomal fragment of the beta globin gene. In the commercial PCRs no consensus and reproducible signals could be detected. The ambivalent results were ascribed to a low and highly degraded DNA content. Since the police were certain of the human origin of the materials, because of the dog’s reaction, mitochondrial DNA sequencing was done. The sequence quality was poor and only a non-valuable sequence was obtained.
* Corresponding author at: Institute of Experimental Medicine, CAU Kiel & University Hospital Schleswig-Holstein, c/o Arnold-Heller-Str. 3, House 28, D24105 Kiel, Germany. Tel.: +49 4315973571; fax: +49 431 5973612. E-mail address: [email protected] (R. Renneberg). 1875-1768/$ – see front matter ß 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.fsigss.2009.08.155
After rethinking the results, as well as the fact that animal hair was found on the blanket, a DNA sample was sent to the Institute of Animal Breeding and Husbandry for additional analysis. Just a few days later the case was closed: the tissue belonged to the placenta of a cow. Due to the fact that the track hounds as well as the chosen PCRs were supposed to be human specific the process of analysis was unnecessarily prolonged. In consequence the used primers, especially mitochondrial primers, were retested for human specificity. 2. Materials and methods DNA from tissue as well as buccal swabs from 13 different species was extracted by an automated extraction (EZ1biorobot, Qiagen). Mitochondrial DNA was amplified by two different mtprimer pairs [4,5]. Cycling parameters were used with adjusted parameters for highly degraded material: 94 8C 5 min followed by 40 cycles of 94 8C 1 min, 57 8C 1 min, 72 8C 1 min for primer set I (expected amplicon length of 279 and 435 bp [4]) and for the primer set II (length of 179 and 434 bp [5]) 95 8C 10 min followed by 40 cycles of 94 8C 1 min, 57 8C 1 min and 72 8C 1 min respectively. Electrophoresis of the PCR products was performed on 2.5% agarose gels and a 6% PAGE. Sequencing was carried out on an ABI Prism310. Next to the sequence process the primer pairs were labelled with fluorescent dye (6-Fam) and applied on the samples, to get the exact fragment-length of PCR products by electrophoresis on the ABI Prism310.
96
R. Renneberg et al. / Forensic Science International: Genetics Supplement Series 2 (2009) 95–96
Table 1 Amplicons detected on an ABI310 after amplification of two mitochondrial DNA fragments with different temperatures and 40 cycles. Species
Amplicons (bp) with 57 8C AnnealT
Amplicons (bp) with 62 8C AnnealT
Homo sapiens Canis familiaris Python sp. Macaca sp. Cervus sp. Ovis aries Bos taurus Sus scrofa Anas sp. Pteropus sp. Pelicanus sp. Castor fiber Felis silvestris Gallus sp.
279, 435 136, 228, 246, 279, 483 156, 160, 166, 177, 279 90, 100, 161, 278, 434, 461 95, 112, 143, 278, 299, 336, 449, 486 278, 300, 482 196, 226, 279, 336, 494 265, 459 158, 266, 279, 306, 532 126, 278 278 438 278 129
279, 435 / / 279 / / / / / / / / / /
/ = no signals detected.
To carry out the optimal conditions for a highly specific PCR a temperature gradient and different numbers of cycles were performed. Additionally, a real-time PCR (which was tested on human specificity before) was carried out on the different samples to exclude the possibility of dealing with contaminant human DNA. In all PCRs a positive control as well as no template controls by a 1:5 ratio were carried along. 3. Results and discussion All samples showed positive results on the agarose gel and PAGE when using the annealing temperatures given in the literature and performing 40 cycles. In most of the samples next to the specific product several other unspecific fragments were obtained. None of the samples could be sequenced in a sufficient manner. The amplicons of the fluorescent labelled primers showed that the several fragments were between 90 and 532 bp long (Table 1). Fragment sizes show that the amplicons might not be derived by mitochondrial DNA; moreover searching with the BLAST tool (NCBI) revealed no complete match of the primers but several possibilities for generating false-positive results due to non-severe PCR parameters and low efficiency of the amplification. Table 1 shows the success rate in enlarging the specificity of the primers by increasing the temperature by 5 8C. Only the phylogenetic closest relative in the sample pool showed positive results due to the fact that the sequence of the primer binding site is very similar. Searching the sequence of Macaca 11 bp of the upper primer and 12 bp of the lower primer matched and matching sites were located at the 50 end and therefore allowed an amplification of the fragment. By reducing the cycle number, the amount of amplified product was reduced in species which are distantly related phylogenetically (birds and reptiles; data not shown). Here the amplification was caused by contaminations. 40 cycles are enough to amplify a single cell over the detection limit of agarose gels and therefore slightest traces of mitochondrial DNA contaminations become visible and might be interpreted as a positive result. These contaminations can originate from PCR tubes and may therefore occur in every PCR but will only become visible if the amount of DNA was high or adequate cycles were carried out to up-amplify the low amount of DNA. In our case real-time PCR proved that no contaminating human DNA was present in the DNA extracts of animal tissues used for these experiments. Therefore the amplified fragments originated from the samples. As a matter of fact false-positive results when analysing unidentified trace materials might occur.
4. Conclusion The analysis of mitochondrial DNA is highly susceptible for contaminations and should therefore be carried out with special cautiousness. But not only environmental conditions as well as contamination prevention are of importance, PCR itself must be ideally optimized to provide specific and highly reliable results. Even when primers are chosen carefully and are human specific, they are applicable on different species if using wrong PCR parameters. The main focus during the design of a new PCR or/and during the adoption of a new system in the lab should be on the specific annealing temperature which might differ from the one quoted by the manufacturer. Moreover, if sequencing results show highly unspecific fragments it is not only a sign of highly degraded DNA material but might also indicate an insufficient amplification. When dealing with material which cannot be determined as human material by morphological, histological or chemical/ physical analysis in general a genetic species determination should be applied first, e.g. [6,7]. Role of funding University research funding. Conflict of interest None. References [1] E. Johnston, C.E. Ames, K.E. Dagnall, J. Foster, B.E. Daniel, Comparison of presumptive blood test kits including hexagon OBTI, J. Forensic Sci. 53 (3) (2008) 687–689. [2] N. von Wurmb-Schwark, A. Preusse-Prange, A. Heinrich, E. Simeoni, T. Bosch, T. Schwark, A new multiplex-PCR comprising autosomal and Y-specific STRs and mitochondrial DNA to analyze highly degraded material, Forensic Sci. Int. Genet. 3 (2) (2009) 96–103. [3] N. von Wurmb-Schwark, T. Schwark, M. Harbeck, M. Oehmichen, A simple duplexPCR to evaluate the DNA quality of anthropological and forensic samples prior short tandem repeat typing, Legal Med. 6 (2004) 80–88. [4] H. Pfeiffer, P. Forster, C. Ortmann, B. Brinkmann, The results of an mtDNA study of 1200 inhabitants of a German village in comparison to other Caucasian databases and its relevance for forensic casework, Int. J. Legal Med. 114 (2001) 169–172. [5] L. Fehren- Schmitz, M. Reindel, E. Tomasto Cagigao, S. Hummel, B. Herrmann, PreColumbian Population Dynamics in Coastal Southern Peru: A Diachronic Investigation of mtDNA Patterns in the Palpa region by Ancient DNA Analysis, Am J Phys Anthropol (2009), doi:10.1002/ajpa.21135. [6] W. Parson, K. Pegoraro, H. Niedersta¨tter, M. Fo¨ger, M. Steinlechner, Species identification by means of the cytochrome b gene, Int. J. Legal Med. 114 (1–2) (2000) 23–28. [7] H.M. Hsieh, H.L. Chiang, L.C. Tsai, S.Y. Lai, N.E. Huang, A. Linacre, J.C. Lee, Cytochrome b gene for species identification of the conservation animals, Forensic Sci. Int. 122 (1) (2001) 7–18.