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Analysis of mtDNA HVIII length heteroplasmy
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Forensic Science International: Genetics Supplement Series 1 (2008) 290–291 www.elsevier.com/locate/FSIGSS
Research article
Analysis of mtDNA HVIII length heteroplasmy A. Nagai *, Y. Bunai Department of Legal Medicine, Graduate School of Medicine, Gifu University, 1-1 Yanagido, Gifu 501-1194, Japan Received 9 September 2007; accepted 11 October 2007
Abstract Length heteroplasmy in the hypervariable region HVIII of mitochondrial DNA (mtDNA) was analyzed by direct sequencing, fragment analysis and cloning. Samples from 19 of 437 unrelated Japanese individuals (4.3%) showed length heteroplasmy in the homopolymeric tract of cytosines (nt 568–573). Every length heteroplasmic sample found in this study was composed predominantly of 9, 10 or 11 cytosines in the homopolymeric tract. # 2008 Published by Elsevier Ireland Ltd. Keywords: Mitochondrial DNA; HVIII; Length heteroplasmy; Japanese
1. Introduction
2.3. Direct sequencing
Mitochondrial length heteroplasmy is variation in the number of bases residing within a homopolymeric stretch in an individual [1,2]. Length heteroplasmy in the third hypervariable region (HVIII) of human mitochondrial DNA (mtDNA) has been observed in homopolymeric tract of cytosines (C-stretch) between nt 568 and nt 573 [3,4]. We analyzed the HVIII length heteroplasmy in a Japanese population using direct sequencing, fragment analysis and cloning in order to identify length variation within the heteroplasmic sample.
The PCR primers fluorescently labelled with Texas Red were used as the sequence primers. The PCR products were directly sequenced from both ends using a SQ5500-S DNA Sequencer (Hitachi Electronics Engineering), and the Thermo Sequenase core sequencing kit or the Thermo Sequenase Primer Cycle Sequencing Kit (both: Amersham) according to the manufacturer’s instructions. The resulting sequence data were compared with the reference sequence described by Anderson et al. [6].
2. Materials and methods 2.1. Samples DNA was extracted from peripheral blood samples of 437 unrelated Japanese individuals living in Gifu Prefecture (central region of Japan) using the phenol–chloroform method or the nucleic acid isolation system QuickGene-810 (Fujifilm).
2.4. Fragment analysis PCR was performed using the primer pair F182/R619 except that R619 was fluorescently labelled with 6-FAM. The PCR products were then digested with restriction enzyme Hae III (Takara Bio). The enzyme-treated PCR products were denatured and separated by capillary electrophoresis using an ABI PRISM 310 Genetic Analyzer (Applied Biosystems). The results were analyzed using GeneScan 3.1.2 (Applied Biosystems).
2.2. PCR Amplification of HVIII region was carried out with primer pair F182/R619 according to Bini et al. [5]. * Corresponding author. Tel.: +81 58 230 6417; fax: +81 58 230 6418. E-mail address: [email protected] (A. Nagai). 1875-1768/$ – see front matter # 2008 Published by Elsevier Ireland Ltd. doi:10.1016/j.fsigss.2007.10.157
2.5. Cloning PCR products were cloned into the pGEM-T Easy Vector System (Promega). Each clone was reamplified with the primer pair F182/R619 and the PCR products were then directly sequenced.
A. Nagai, Y. Bunai / Forensic Science International: Genetics Supplement Series 1 (2008) 290–291
291
Table 1 Distribution of length variants in HVIII length heteroplasmic samples Sample
A B C D E F G
Predominant sequence in fragment analysis
C10 C10 C11 C9 C10 C11 C10
3. Results and discussion The mtDNA HVIII region of 437 unrelated Japanese individuals was amplified and directly sequenced. A total of 19 samples (4.3%) showed length heteroplasmy in the homopolymeric Cstretch (nt 568–573). To confirm the presence of length heteroplasmy, fragment analysis was performed for the length heteroplasmic samples. In the electropherograms, all of the length heteroplasmic samples showed a single peak. The fragment size of each length heteroplasmic sample was 322, 323 or 324 bp. Based on the fragment size (319 bp) of a homoplasmic sample (sequence of length C6 at the homopolymeric C-stretch), the sizes of the length heteroplasmic samples, 322, 323 and 324 bp, corresponded to sequences of length C9, C10 and C11, respectively. Therefore, it was suggested that the predominant sequence in the length heteroplasmy found in this study was C9, C10 or C11. To investigate length variation within the length heteroplasmy, cloning analysis was performed for 7 out of 19 length heteroplasmic samples. The results showed that the predominant sequences suggested by the fragment analysis were the major elements of the length heteroplasmic samples found in this study (Table 1). The analysis of length heteroplasmy in the mtDNA HVIII region will increase the usefulness of this region in the forensic identification.
Variants of C residues
Total clones
C9
C10
C11
C12
1 1 0 13 1 0 1
5 6 2 1 7 0 6
3 2 8 0 4 7 2
0 1 3 0 2 3 0
9 10 13 14 14 10 9
Conflict of interest None.
References [1] A. Carracedo, et al., DNA Commission of the International Society for Forensic Genetics: guidelines for mitochondrial DNA typing, Forensic Sci. Int. 110 (2000) 79–85. [2] J.E.B. Stewart, et al., Length variation in HV2 of the human mitochondrial DNA control region, J. Forensic Sci. 46 (2001) 862–870. [3] H.Y. Lee, et al., Mitochondrial DNA control region sequences in Koreans: identification of useful variable sites and phylogenetic analysis for mtDNA data quality control, Int. J. Legal Med. 120 (2006) 5–14. [4] A. Nagai, I. Nakamura, Y. Bunai, Analysis of the HVI, HVII and HVIII regions of mtDNA in 400 unrelated Japanese, in: A. Amorim, F. CorteReal, N. Morling (Eds.), Progress in Forensic Genetics, vol. 11, Elsevier, Amsterdam, 2006, pp. 139–141. [5] C. Bini, et al., Population data of mitochondrial DNA region HVIII in 150 individuals from Bologna (Italy), in: B. Brinkmann, A. Carracedo (Eds.), Progress in Forensic Genetics, vol. 9, Elsevier, Amsterdam, 2003, pp. 525– 528. [6] S. Anderson, et al., Sequence and organization of the human mitochondrial genome, Nature 290 (1981) 457–465.
Forensic Science International: Genetics Supplement Series 1 (2008) 290–291 www.elsevier.com/locate/FSIGSS
Research article
Analysis of mtDNA HVIII length heteroplasmy A. Nagai *, Y. Bunai Department of Legal Medicine, Graduate School of Medicine, Gifu University, 1-1 Yanagido, Gifu 501-1194, Japan Received 9 September 2007; accepted 11 October 2007
Abstract Length heteroplasmy in the hypervariable region HVIII of mitochondrial DNA (mtDNA) was analyzed by direct sequencing, fragment analysis and cloning. Samples from 19 of 437 unrelated Japanese individuals (4.3%) showed length heteroplasmy in the homopolymeric tract of cytosines (nt 568–573). Every length heteroplasmic sample found in this study was composed predominantly of 9, 10 or 11 cytosines in the homopolymeric tract. # 2008 Published by Elsevier Ireland Ltd. Keywords: Mitochondrial DNA; HVIII; Length heteroplasmy; Japanese
1. Introduction
2.3. Direct sequencing
Mitochondrial length heteroplasmy is variation in the number of bases residing within a homopolymeric stretch in an individual [1,2]. Length heteroplasmy in the third hypervariable region (HVIII) of human mitochondrial DNA (mtDNA) has been observed in homopolymeric tract of cytosines (C-stretch) between nt 568 and nt 573 [3,4]. We analyzed the HVIII length heteroplasmy in a Japanese population using direct sequencing, fragment analysis and cloning in order to identify length variation within the heteroplasmic sample.
The PCR primers fluorescently labelled with Texas Red were used as the sequence primers. The PCR products were directly sequenced from both ends using a SQ5500-S DNA Sequencer (Hitachi Electronics Engineering), and the Thermo Sequenase core sequencing kit or the Thermo Sequenase Primer Cycle Sequencing Kit (both: Amersham) according to the manufacturer’s instructions. The resulting sequence data were compared with the reference sequence described by Anderson et al. [6].
2. Materials and methods 2.1. Samples DNA was extracted from peripheral blood samples of 437 unrelated Japanese individuals living in Gifu Prefecture (central region of Japan) using the phenol–chloroform method or the nucleic acid isolation system QuickGene-810 (Fujifilm).
2.4. Fragment analysis PCR was performed using the primer pair F182/R619 except that R619 was fluorescently labelled with 6-FAM. The PCR products were then digested with restriction enzyme Hae III (Takara Bio). The enzyme-treated PCR products were denatured and separated by capillary electrophoresis using an ABI PRISM 310 Genetic Analyzer (Applied Biosystems). The results were analyzed using GeneScan 3.1.2 (Applied Biosystems).
2.2. PCR Amplification of HVIII region was carried out with primer pair F182/R619 according to Bini et al. [5]. * Corresponding author. Tel.: +81 58 230 6417; fax: +81 58 230 6418. E-mail address: [email protected] (A. Nagai). 1875-1768/$ – see front matter # 2008 Published by Elsevier Ireland Ltd. doi:10.1016/j.fsigss.2007.10.157
2.5. Cloning PCR products were cloned into the pGEM-T Easy Vector System (Promega). Each clone was reamplified with the primer pair F182/R619 and the PCR products were then directly sequenced.
A. Nagai, Y. Bunai / Forensic Science International: Genetics Supplement Series 1 (2008) 290–291
291
Table 1 Distribution of length variants in HVIII length heteroplasmic samples Sample
A B C D E F G
Predominant sequence in fragment analysis
C10 C10 C11 C9 C10 C11 C10
3. Results and discussion The mtDNA HVIII region of 437 unrelated Japanese individuals was amplified and directly sequenced. A total of 19 samples (4.3%) showed length heteroplasmy in the homopolymeric Cstretch (nt 568–573). To confirm the presence of length heteroplasmy, fragment analysis was performed for the length heteroplasmic samples. In the electropherograms, all of the length heteroplasmic samples showed a single peak. The fragment size of each length heteroplasmic sample was 322, 323 or 324 bp. Based on the fragment size (319 bp) of a homoplasmic sample (sequence of length C6 at the homopolymeric C-stretch), the sizes of the length heteroplasmic samples, 322, 323 and 324 bp, corresponded to sequences of length C9, C10 and C11, respectively. Therefore, it was suggested that the predominant sequence in the length heteroplasmy found in this study was C9, C10 or C11. To investigate length variation within the length heteroplasmy, cloning analysis was performed for 7 out of 19 length heteroplasmic samples. The results showed that the predominant sequences suggested by the fragment analysis were the major elements of the length heteroplasmic samples found in this study (Table 1). The analysis of length heteroplasmy in the mtDNA HVIII region will increase the usefulness of this region in the forensic identification.
Variants of C residues
Total clones
C9
C10
C11
C12
1 1 0 13 1 0 1
5 6 2 1 7 0 6
3 2 8 0 4 7 2
0 1 3 0 2 3 0
9 10 13 14 14 10 9
Conflict of interest None.
References [1] A. Carracedo, et al., DNA Commission of the International Society for Forensic Genetics: guidelines for mitochondrial DNA typing, Forensic Sci. Int. 110 (2000) 79–85. [2] J.E.B. Stewart, et al., Length variation in HV2 of the human mitochondrial DNA control region, J. Forensic Sci. 46 (2001) 862–870. [3] H.Y. Lee, et al., Mitochondrial DNA control region sequences in Koreans: identification of useful variable sites and phylogenetic analysis for mtDNA data quality control, Int. J. Legal Med. 120 (2006) 5–14. [4] A. Nagai, I. Nakamura, Y. Bunai, Analysis of the HVI, HVII and HVIII regions of mtDNA in 400 unrelated Japanese, in: A. Amorim, F. CorteReal, N. Morling (Eds.), Progress in Forensic Genetics, vol. 11, Elsevier, Amsterdam, 2006, pp. 139–141. [5] C. Bini, et al., Population data of mitochondrial DNA region HVIII in 150 individuals from Bologna (Italy), in: B. Brinkmann, A. Carracedo (Eds.), Progress in Forensic Genetics, vol. 9, Elsevier, Amsterdam, 2003, pp. 525– 528. [6] S. Anderson, et al., Sequence and organization of the human mitochondrial genome, Nature 290 (1981) 457–465.