MtDNA Sequence Analysis Using Capillary Electrophoresis and Its Application to the Analysis of MtDNA in Hair

Transcription

1 鑑識科学,7(2), (2003) 123 Original Article MtDNA Sequence Analysis Using Capillary Electrophoresis and Its Application to the Analysis of MtDNA in Hair Kazumasa Sekiguchi, Kazuhiko Imaizumi, Hideaki Matsuda, Natsuko Mizuno, Kanako Yoshida, Hiroaki Senju, Hajime Sato and Kentaro Kasai National Research Institute of Police Science 6 3 1, Kashiwanoha, Kashiwa, Chiba , Japan (Received 11 June 2002; accepted 12 August 2002) A procedure for the analysis of mitochondrial DNA (mtdna) using capillary electrophoresis instead of former gel-based methods is described. The procedure requires less manual manipulation in terms of electrophoresis and, therefore, reduces the chance of either human- or gel-related failures. Thus, the method is suitable for performing mtdna typing from limited amounts of forensic samples. We also performed mtdna typing of hair samples using this method, and were successful in typing from both hair roots and hair shafts as well as saliva and nail samples. The amount of PCR product indicated that the amount of mtdna in the tip side of the hair shaft was less than that in the root side. However, one hair sample showed equal amounts of PCR products in both the tip and the root side. For the analysis of a sample derived from an individual with heteroplasmic mtdna, the proportions of heteroplasmy from the saliva and nail samples were dišerent from those from hair samples. For analyses of hairs from the same individual, each region of the hair showed dišerent proportions of heteroplasmy and the results indicated the possibility of the dišerent sequences in the same hair sample. Therefore, mtdna analysis of hair samples will require additional investigation of procedures for heteroplasmic mtdna. These results strongly suggest that the application of the developed method for hair samples will require careful treatment of the samples and a rigorous analysis of the results. Key words: Mitochondrial DNA, Control region, Hair DNA; Heteroplasmy, Capillary electrophoresis Introduction Human mitochondrial DNA (mtdna) has been completely sequenced. The control region in the major non-coding portion is located around the replication origin 1). For some forensic samples such as bone 2) and hair 3), only degraded DNA can be extracted and, as a result, it is di cult to perform DNA typing of the nuclear loci. However, mtdna analysis can be used for such samples because mtdna exists as numerous molecules in a single cell in contrast to other nuclear DNA 4,5). Numerous polymorphisms found in the two hypervariable region (HV1 and HV2) of the control region have been reported 6 11) and are used as an informative tool for the identiˆcation of individuals from forensic samples such as human hairs 12), bones 13),andteeth 14).

2 124 Kazumasa Sekiguchi et al. Our previous study also reported on polymorphisms in this region in the Japanese population 15). Since our previous method was laborious and required considerable experience, a simpler systematic method using Catalyst 877 and a PRISM310 sequencer (Applied Biosystems) was developed and is described herein. We also applied this method to the determination of mtdna sequences from hairs and discuss problems encountered in the identiˆcation of human hairs. Material and Methods 1. Bloodstains, saliva stains, nails and hairs Bloodstains and saliva stain samples were collected from unrelated Japanese individuals and were preserved in a -80 C freezer until used for DNA extraction. Saliva (cotton swabs), nails andhairsamples(morethan10cminlength) were collected from a Japanese female and a bloodstain sample was also collected from the same subject. 2. DNA extraction and quantitation Bloodstains and saliva samples were incubated for 1 hour at 70 C with 100 mg/ml of Proteinase K and 1 SDS in TNE bušer (100 mm Tris-Cl ph 8.0, 500 mm NaCl, 10 mm EDTA). The DNA was puriˆed by the phenol/chloroform/isoamylalcohol procedure, precipitated with ethanol and resuspended in TE bušer (10 mm Tris-Cl ph 8.0, 1 mm EDTA) 16,17). DNA concentrations were determined by absorbance measurements at 260 nm. The sensitivity study was performed with serial dilution of DNA solutions by TE. After serial washing procedure of Terg-A- Zyme (Alconox), water, and ethanol, each hair was cut into a 5 mm section, which included the root, a 5 cm section of hair shaft proximal to the 5 mm section, and a 5 cm section including the hair tip (Table 1). Each section was incubated for1hourat55 C in40mmdtt,100mg/ml Proteinase K and 1 SDS in TNE bušer. The DNA was puriˆed by the phenol/chloroform/ isoamylalcohol procedure, concentrated with Microcon 100 Centrifugal Filter Units and diluted to a ˆnal volume of 15 ml intebušer 3). DNA from nail samples were extracted using the same procedure as was used for hair samples. 3. Primers Seven PCR primers were designed for the ampliˆcation of both the HV1 and HV2 regions (Figs. 1 and 2). Five sets of primers can be used to amplify and determine the entire sequence of HV1 and HV2 (Table 2). Three of these (HV1 B, HV1 C andhv2 E) are for DNA samples which contain the ``C-stretch'' region 18).Ineach forward PCR primer (F), the -21M13 primer sequence is attached at the 5 end,andineach reverse PCR primer (R), the M13REV primer sequence is attached at the 5 endsothateither the DyePrimer or DyeTerminator method can be used for sequencing. 4. PCR The ampliˆcations were conducted using 1ng of DNA from the bloodstain samples, and a 2 ml aliquot of the DNA suspension from the hair samples. PCR was performed in a 25 ml volume containing 1.25U of AmpliTaq GOLD DNA polymerase, 1x PCR GOLD BuŠer, 200 mm dntps, 0.02 Bovine Serum Albumin and 0.5 mm of the forward and reverse primers. Thermal cycling was performed using a GeneAmp PCR system 9700 starting with 9 min at 95 C, followed by 33 cycles of 45 sec at 95 C, 30 sec at 60 C witha50 ramp and 2 min at Hair Length (cm) Table 1 Status Sample names of hairs used in this experiment. 5 mm including hairbulb Sampling regions 5cmfromhair root except hairbulb 5cmof hairshaft 5 cm from hair end Telogen H1 1 H1 2 H Telogen H2 1 H2 2 H Anagen H3 1 H3 2 H3 3 H3 4

3 MtDNA Sequence Analysis Using Capillary Electrophoresis and Its Application to the Analysis of MtDNA in Hair 125 Fig. 1 Scheme showing the position of PCR primers and the regions for ampliˆcation of the control region of mtdna. Fig. 2 Primers used for mtdna ampliˆcation. Lower case letters represent sequences of either M13FWD or M13REV supplied by ABD. Upper case letters represent the sequences of the mtd- NA control region. 72 C. A 5 ml aliquot of the PCR product was runona2 NusieveMEagarosegelinTAE bušer and visualized by ethidium bromide staining to conˆrm the quality and quantity of the product. According to the recommendations of the DNA Commission of the International Society for Forensic Genetics (ISFG) 19), the possibility of contamination was monitored by simultaneously extracting, purifying and amplifying reagent blanks and negative controls. No DNA was detectable from the reagent blanks or the negative controls as evidenced by electrophoresis on an agarose gel. 5. Puriˆcation of PCR products Table 2 Combinations of primers used for the ampliˆcation of each region. Regions Primer Primer Sets Sets Forward Reverse A L15997F H16401R HV1 B L15997F H16174R C L16208F H16401R HV2 D L00029F H00438R E L00029F H00290R The PCR products were puriˆed and concentrated to 10~30 ml by means of a Microcon-100 device. The concentration of the puriˆed PCR products was then determined by an optical density measurement at 260 nm. 6. Sequencing Puriˆed PCR products were sequenced using either BigDye TM primer Cycle Sequencing Kits or BigDye TM Terminator Cycle Sequencing Kits (Applied Biosystems). Sequencing reactions were performed in both directions to conˆrm the sequence. These reactions were also performed using the Catalyst 877 Integrated Thermal Cycler (Applied Biosystems). Sequence data were obtained on a PRISM 310 genetic analyzer (Applied Biosystems) using POP6. Sequence data were analyzed with the Sequencing Analysis Software 3.3 (Applied Biosystems) and

4 126 Kazumasa Sekiguchi et al. PCR Sequencing Table 3 Comparison of previously described procedures and the present method. Procedure This method using PRISM 310 Previous method using Li COR Sequencer PCR ampliˆcation 1 step 2 steps Total PCR time 3 hours 6 hours PCR sensitivity 1 pg~10 pg 100 fg~1 pg Puriˆcation of PCR product Easy but slightly expensive Laborious but relatively cheap Reaction Dye Primer Dye Terminator Dye Primer Puriˆcation of Sequencing product Required None Gel Preparation None Required Sample loading Automatically loading Manually loading Time of Electrophoresis 50 minutes per one sample 6 hours per 16 samples Analysis Automatically Semi automatically comparisons were made using Sequence Navigator Software 1.01 (Applied Biosystems). Results 1. Sensitivity and comparison of procedures between the previous and present methods The sensitivity of the PCR ampliˆcation of the newly designed PCR primers was veriˆed. The sensitivity varied slightly among primer sets, but su cient PCR products were obtained from the 10 pg template DNA for all primer sets except HV1 B. However, these sensitivities dišered by a factor of 10 to 100 times among individuals, indicating that the amount of mtdna varied considerably between individuals or each sample. Comparisons of procedures between previously reported methods and this study are shown in Table 3. The PCR sensitivity was lower than that found for the previous nested PCR method, but the amount required for the PCR was less than that reported for the previous method and the over-all procedures, which eliminated the need for gel preparation and sample application were less laborious. Furthermore, the DyeTerminator method as well as DyePrimer method can be used for the Fig. 3 PCR ampliˆcation of the HV2 D region from various regions of hair. Lanes (H1 1 through H3 4) are described in Table 1. sequencing reaction and it was possible to conˆrm the proportion of heteroplasmy in both methods. 2. MtDNA sequence from hair, nail and saliva PCR ampliˆcations were performed from 10 portions of 3 hair samples, as shown in Table 1. The results of the ampliˆcation of the HV2 D region are shown in Fig 3. Other regions were also examined and similar results were obtained. In a comparison of the amount of PCR

5 MtDNA Sequence Analysis Using Capillary Electrophoresis and Its Application to the Analysis of MtDNA in Hair 127 ampliˆcation within the same hair sample, the hair root region (H1 1, H2 1, H3 1) was ampliˆed to a greater extent than the hair shaft regions, even though the root region was shorter than the shaft region. The e ciencies of ampliˆcation varied among hairs. The hair tip regions in hair 1 and hair 2 showed less ampliˆcation than the hair shafts proximal to the root, whereas all the regions in hair 3 showed almostthesamedegreeofampliˆcation. After the sequence analysis, this person contained an Adenine (A): Guanine (G) heteroplasmy at position 234 in the HV2 region (Fig. 4). Although the proportion of heteroplasmy was slightly dišerent, this heteroplasmy was observed in both the DyePrimer and DyeTerminator sequencing method and in both directions. Sequencing reactions and analyses of the hair samples (H1 1 Fig. 4 Chromatograms of the HV2 region of H2 1 (hair root) using two sequencing methods in both directions. The position indicated by an arrow shows the G/A heteroplasmy in nucleotide position 234. Table 4 The nucleotide at position 234 in HV2, as determined with the Sequence Analysis Software. Sample Number of Sequence reactions Numbersofresults in which each nucleotide was displayed by Sequence Analysis software a) Average Proportion of 234A b) A G N H H H H H H H H H H N N N S a) Total numbers of the results shown this table are less than total numbers of sequence reactions because of failures of either the electrophoresis or the sequencing reactions. b) The proportions of heteroplasmy were estimated by averaging the percentage of the height of the A peak compared to that of the G peak at position 234 in all electropherograms from each sample.

6 128 Kazumasa Sekiguchi et al. through H3 4) were repeatedly conducted for the HV2 region (HV2 D andhv2 E) including position 234. After an analysis using the Sequence Analysis software, the A:G heteroplasmy at position 234 was typed to be either A, G, or N in each sequence reaction even for the same hair (Table 4). The proportions of heteroplasmy were estimated by averaging the percentage of the height of the A peak at position 234 compared to that of the G peak in all electropherograms obtained from two sequencing chemistries and from both directions (Table 4 and Fig. 5). The proportion of A:G heteroplasmy among hairs indicated a wide variation. Hair root regions (H1 1, H2 1 and H3 1) and two of the hair shaft regions (H3 3 and H3 4) ranged from about 35 Ato53 A. Three hair shaft regions (H1 2, H2 3 andh3 2) were in the range of 12 Ato24 A, whereas two hair shaft regions (H1 3 andh2 2) were found to be about 80 A and 84 A. DNA extracted from nail samples (N1, N2 and N3) and a saliva sample (S) were also sequenced and the average proportions are shown in Table 4 and Fig. 5. The proportion of heteroplasmy observed in nails (N1, N2 and N3) wasintherangeof51 Ato75 Aandthatin the saliva sample (S) was approximately 54 A. Discussion In this study, we report on the sequencing of the mtdna control regions, HV1 (15,998 16,400) and HV2 (30 437). These regions are su ciently wide for comparing with other mtdna databases 11). We used a ``tagged primer'' for mtdna ampliˆcation because this permits the DyePrimer method as well as the DyeTerminator method to be used for sequencing. We used dišerent tags for a forward or reverse primer in order to conduct both forward and reverse strand sequencing reactions from a single PCR ampliˆcation 19,20).Atˆrst,we think there was a signiˆcantly dišerent result obtained from the DyePrimer method and the DyeTerminator method, but, in this study, the level of detection of heteroplasmy was similar for both methods in this position. As shown in Table 4, the result of an analysis using Sequence Analysis and Sequence Navigator software did not always perform base calling as heteroplasmy (N), which was only displayed when the height of two heteroplasmic peaks were almost the same. In most cases, only a major peak was detected and reported even though a small minor peak was obviously present in the electropherogram. Although BigDye Terminators give much more even peaks than other kits, uneven peak heights are still found in some speciˆc sequence contexts because Fig. 5 Proportions of mtdna heteroplasmy observed in various positions of hairs, nails and saliva samples Positions, H1 1 through H3 4, are shown in Table 1.

7 MtDNA Sequence Analysis Using Capillary Electrophoresis and Its Application to the Analysis of MtDNA in Hair 129 of the character of the AmpliTaq enzyme 21). Uneven peak heights depending on sequence contexts may cause the dišerence of the proportion of heteroplasmy. This indicates that analyses of mtdna heteroplasmy should be checked both by sequencing repeatedly in both directions and by visual inspection of the electropherograms. Length heteroplasmy has been reported within one individual 18,22,23), and variable proportions of heteroplasmy have also been observed in hair roots within a single individual 24,25). Our study also indicates variations in the proportions of heteroplasmy, which were observed in hairs, nails and saliva from a single individual. Even within the same hair, the proportions of heteroplasmy were dišerent at a given position. These results suggest that if one individual has mtdna heteroplasmy either in the C-stretch region or other regions, his/her hair samples may have dišerent proportions of heteroplasmy for blood or saliva samples as well as between his/her individual hairs. Therefore, for forensic human identiˆcation using mtdna sequence polymorphisms of trace samples such as hairs and nails, attention must be focused on the possibility of heteroplasmy, and as many samples as possible from one individual should be examined. References 1) Anderson, S., Bankier A. T., Barrell B. G., de Bruijn M. H., Coulson A. R., Drouin J., Eperon I. C., Nierlich D. P., Roe B. A., Sanger F., Schreier P. H., Smith A. J., Staden R. and Young I. G.: Sequence and organization of the human mitochondrial genome. Nature, 290, (1981). 2) Imaizumi, K., Matsuda H., Kubota S., Miyasaka S. and Yoshino M.: The EŠects of Decalciˆcation and Glass Beads Treatment on DNA Typings from Bone Samples. Rep. Natl. Res. Inst. Police Sci, 51, (1998). 3) Matsuda, H., Sekiguchi K., Kasai K., Yoshino M. and Seta S.: Evaluation of Various Methods of DNA Analysis for Hair Samples. Jpn. J. Sci. Tech. Iden., 2, (1997). 4) Robin, E. D. and Wong R.: Mitochondrial DNA molecules and virtual number of mitochondria per cell in mammalian cells. J. Cell. Physiol., 136, (1988). 5) Wilson,M.R.,DiZinnoJ.A.,Polanskey D., Replogle J. and Budowle B.: Validation of mitochondrial DNA sequencing for forensic casework analysis. Int. J. Legal Med., 108, (1995). 6) Sullivan, K. M., Hopgood R. and Gill P.: Identiˆcation of human remains by ampliˆcation and automated sequencing of mitochondrial DNA. Int. J. Legal Med., 105, (1992). 7) Piercy, R., Sullivan K. M., Benson N. and Gill P.: The application of mitochondrial DNA typing to the study of white Caucasian genetic identiˆcation. Int. J. Legal Med., 106, (1993). 8) Mountain, J. L., Hebert J. M., Bhattacharyya S., Underhill P. A., Ottolenghi C., Gadgil M. and Cavalli-Sforza L. L.: Demographic history of India and mtdna-sequence diversity. Am. J. Hum. Genet., 56, (1995). 9) Lee, S. D., Shin C. H., Kim K. B., Lee Y. S. and Lee J. B.: Sequence variation of mitochondrial DNA control region in Koreans. Forensic Sci. Int., 87, (1997). 10) Richards, M., Corte-Real H., Forster P., Macaulay V., Wilkinson-Herbots H., Demaine A., Papiha S., Hedges R., Bandelt H. J. and Sykes B.: Paleolithic and neolithic lineages in the European mitochondrial gene pool. Am. J. Hum. Genet., 59, (1996). 11) Budowle, B., Wilson M. R., DiZinno J. A.,StauŠerC.,FasanoM.A.,HollandM. M. and Monson K. L.: Mitochondrial DNA regions HVI and HVII population data. Forensic Sci. Int., 103, (1999). 12) Wilson, M. R., Polanskey D., Butler J., DiZinno J. A., Replogle J. and Budowle B.: Extraction, PCR ampliˆcation and

8 130 Kazumasa Sekiguchi et al. sequencing of mitochondrial DNA from human hair shafts. BioTechniques, 18, (1995). 13) Holland, M. M., Fisher D. L., Mitchell L. G., Rodriquez W. C., Canik J. J., Merril C. R. and Weedn V. W.: Mitochondrial DNA sequence analysis of human skeletal remains: identiˆcation of remains from the Vietnam War. J. Forensic Sci., 38, (1993). 14) Ginther, C., Issel-Tarver L. and King M. C.: Identifying individuals by sequencing mitochondrial DNA from teeth. Nat. Genet., 2, (1992). 15) Sekiguchi, K., Imaizumi K., Fujii K., Senju H., Mizuno N., Sakai I., Kasai K., Sato H. and Seta S.: Sequence Polymorphisms of the Control Region of Human Mitochondrial DNA in Japanese Population. Jpn. J. Sci. Tech. Iden., 2,33 40 (1997). 16) Sakai, I., Kasai K., Yoshida K. and Mukoyama H.: An improved method for DNA isolation from human bloodstain and typing of the isolated DNA using single locus VNTR probe. Rep. Natl. Res. Inst. Police Sci., 44, (1991). 17) Sekiguchi, K., Kasai K. and Sato H.: Evaluation of Various DNA Extractino Kits for Bloodstains. [II]. Rep. Natl. Res. Inst. Police Sci., 52, (1999). 18) Bendall, K. E. and Sykes B. C.: Length heteroplasmy in the ˆrst hypervariable segment of the human mtdna control region. Am. J. Hum. Genet., 57, (1995). 19) Tully, G., Bar W., Brinkmann B., Carracedo A., Gill P., Morling N., Parson W. and Schneider P.: Considerations by the European DNA proˆling (EDNAP) group on the working practices, nomenclature and interpretation of mitochondrial DNA proˆles. Forensic Sci. Int., 124, (2001). 20) Comparative PCR Sequencing-A Guide to Sequencing-Based Mutation Detection, Perkin Elmer Corpoation, Foster City, CA (1995) 21) Zakeri, H., Amparo G., Chen S. M., Spurgeon S. and Kwok P. Y.: Peak height pattern in dichloro-rhodamine and energy transfer dye terminator sequencing. BioTechniques, 25, , (1998). 22) Stewart, J. E., Fisher C. L., Aagaard P. J., Wilson M. R., Isenberg A. R., Polanskey D., Pokorak E., DiZinno J. A. and Budowle B.: Length variation in HV2 of the human mitochondrial DNA control region. J. Forensic Sci., 46, (2001). 23) Salas, A., Lareu M. V. and Carracedo A.: Heteroplasmy in mtdna and the weight of evidence in forensic mtdna analysis: a case report. Int. J. Legal Med., 114, (2001). 24) Bendall, K. E., Macaulay V. A. and Sykes B. C.: Variable levels of a heteroplasmic point mutation in individual hair roots. Am. J. Hum. Genet., 61, (1997). 25) Wilson,M.R.,PolanskeyD.,ReplogleJ., DiZinno J. A. and Budowle B.: A family exhibiting heteroplasmy in the human mitochondrial DNA control region reveals both somatic mosaicism and pronounced segregation of mitotypes. Hum. Genet., 100, (1997).