- Email: [email protected]
Usefulness of deoxyribonuclease I (DNase I) polymorphism for individualization from small aged urine stains
Legal Medicine 5 (2003) 105–107 www.elsevier.com/locate/legalmed
Brief communication
Usefulness of deoxyribonuclease I (DNase I) polymorphism for individualization from small aged urine stains Toshihiro Yasudaa, Haruo Takeshitab, Misuzu Uekia, Tamiko Nakajimab, Kouichi Mogib, Yasushi Kanekob, Reiko Iidac, Koichiro Kishib,* a
b
Department of Biology, Fukui Medical University, Fukui 910-1193, Japan Department of Legal Medicine, Gunma University School of Medicine, Gunma 371-8511, Japan c Department of Legal Medicine, Fukui Medical University, Fukui 910-1193, Japan
Received 21 February 2003; received in revised form 18 March 2003; accepted 20 March 2003
Abstract We devised a procedure that combines a simple extraction method, isoelectric focusing and activity staining using the dried agarose film overlay method, for deoxyribonuclease I (DNase I) typing from aged urine stains. DNase I types were determined without difficulty from urine stains kept at room temperature for 3 months or more in all of the samples tested. The amounts of urine stains required for typing after 3 months of storage were estimated to be equivalent to 60 – 120 ml of liquid urine. Therefore, considering that useful PCR-based DNA typing has not yet been developed for urine stains, DNase I polymorphism could be considered the first biochemical marker found to be well suited for individualization from small aged urine stains. q 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Deoxyribonuclease I polymorphism; Isoelectric focusing; Stains; Urine; Forensic individualization
1. Introduction Deoxyribonuclease I (DNase I; E.C.3.1.21.1) activity is distributed widely in human body fluids, and extremely high levels of the activity are detectable in urine [1,2]. Genetic polymorphisms including biochemical markers [3] and DNA markers [4,5] have been identified and employed for the individualization of liquid urine samples. However, most urine samples encountered by practicing forensic biologists in casework situations are dried stains, and genetic markers suitable for personal identification * Corresponding author. Tel.: þ81-27-220-8030; fax: þ 81-27220-8035. E-mail address: [email protected] (K. Kishi).
from such samples are extremely limited: useful PCRbased DNA typing method has not yet been developed for urine stains. Therefore, it is essential to devise a new and effective system of discrimination from aged urine stains. In this communication, we describe a novel method for DNase I phenotyping from small aged urine stains.
2. Materials and methods 2.1. Biological samples Urine samples were collected from healthy Japanese volunteers including laboratory workers and students,
1344-6223/03/$ - see front matter q 2003 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S1344-6223(03)00049-X
106
T. Yasuda et al. / Legal Medicine 5 (2003) 105–107
all of whom had provided written informed consent. The samples were concentrated, dialyzed and lyophilized as a control material according to a method described previously [6]. A 0.05% w/v solution of the lyophilized materials, corresponding to fivefold concentrated urine, was used for DNase I phenotyping. Artificial urine stains of three common DNase I phenotypes (1, 1 – 2 and 2) were prepared on both filter paper (No. 526, Toyo Roshi Kaisha Ltd., Tokyo, Japan) and two kinds of cloth made of cotton and a mixture of cotton and polyester fiber. A small sheet of each material measuring 2.5 £ 2.5 cm, on which 1 ml of urine freshly collected from each individual was dropped and absorbed, was air-dried for 1 day at room temperature, and stored either at room temperature or at 4 8C, until required for DNase I phenotyping. 2.2. Extraction of DNase I from urine stains Extraction of DNase I from urine stains was performed essentially according to the microextraction method reported previously [7,8], except that 0.1 M MES buffer (pH 6.5) containing 10 mM MgCl2 and 0.1 mM CaCl2 was used as the extraction buffer. The extract recovered from each urine-stained sheet was dialyzed against 0.05% w/v glycine and then lyophilized. The resulting materials were dissolved in 50 ml 0.05% w/v BSA and used for subsequent analysis. 2.3. DNase I phenotyping Isoelectric focusing on a thin layer of polyacrylamide gel (IEF-PAGE) for DNase I phenotyping was performed by a method described previously [6]. Samples for phenotyping were treated with an equal volume of 5 unit/ml Clostridium perfringens neuraminidase (type V, Sigma, St. Louis, MO, USA) overnight at 4 8C before electrophoresis. Following the electrophoretic run, visualization of DNase I was achieved using the dried agarose film overlay (DAFO) method [6].
3. Results and discussion In our previous studies on successful DNase I phenotyping from saliva stains [9] and used socks [8], incorporation of a clean-up step using
Phenyl-Sepharose CL-6B column chromatography prior to electrophoresis was indispensable in order to avoid formation of faint and indistinct bands during IEF-PAGE analysis. However, the extraction procedure without such a clean-up step allowed DNase I to be phenotyped from aged urine stains without difficulty, perhaps due to the higher content of DNase I in urine than in other body fluids [2]. Furthermore, although detergent, BSA or urea had been added to the extraction solution in order to increase the efficiency of DNase I extraction from other body fluid stains, even the buffer optimal for DNase I reaction without addition of such chemicals was found to be effective for DNase I extraction from urine stains: the recovery of DNase I activity extracted from each of the urine stains kept at room temperature for 1, 2, 3 and 4 weeks was estimated to be 90 ^ 7%, 75 ^ 19%, 74 ^ 7% and 65 ^ 10%, respectively, compared with that in freshly collected liquid urine. Also, addition of detergents, such as NP-40 suitable for DNase I extraction from saliva stains [9], to the extraction solution had no favorable effect on extraction of DNase I from the aged urine stains. Therefore, the simple extraction procedure without a clean-up step as described in Section 2 was employed for DNase I typing from urine stains throughout this study. All the stains kept at room temperature or 48C for up to three months or more were correctly typed without false interpretation. The amount of urine stain required for DNase I phenotyping was estimated to be a 0.4– 0.8 cm2 spot on filter paper (equivalent to 60 –120 ml of liquid urine) after 3 months of storage, irrespective of the storage temperature. These amounts of urine are sufficient to perform DNase I phenotyping from items of evidence submitted in criminal investigations. Furthermore, a longer incubation with the DAFO sheet or a slight elevation of the incubation temperature permits DNase I to be typed from much smaller and older aged urine stains. Since urine samples recovered from crime scenes are commonly encountered as stains dried on cloth, the effect of materials on which liquid urine is absorbed on DNase I phenotyping from these stains should be examined. Phenotyping from two kinds of urine-stained cloth made of cotton and a mixture of cotton and polyester fiber could be performed in the same manner as for urine-stained filter paper. Therefore, it could be concluded that success or failure of
T. Yasuda et al. / Legal Medicine 5 (2003) 105–107
DNase I phenotyping from urine stains does not depend on the material components of the cloth on which the urine has been deposited. Few genetic markers are applicable for personal identification from dried urine stains, which are commonly found at crime scenes; only ABO phenotyping is used in practice, and is useful only if the stains have been deposited by secretor-type persons [10]. Since high levels of DNase I activity were found to be conserved on aged urine stains, urinary DNA may be too badly degraded during storage of the stains to be typed. In fact, correct STR typing could not be performed from a liquid urine samples kept for only 1 week at room temperature [5]. Furthermore, the low content and relatively labile nature of the genetic biochemical markers characterized in urine have previously hindered the application of these markers to individualization of urine stains. On the other hand, the favorable distribution of the DNase I gene frequencies [1], the highly stable nature of the DNase I protein [11] and the high levels of its activity in human urine allow DNase I polymorphism to be well suited for this purpose. Availability of a highly sensitive phenotyping method employing DAFO activity staining has enabled DNase I typing to be performed on aged body fluid stains including urine stains. Therefore, it is concluded that DNase I polymorphism is the first genetic marker to be found effective for forensic individualization of urine stains. Biochemical markers like DNase I polymorphism, which can be detected by conventional, but well approved and established methods, can be useful for forensic investigations of criminal materials if they bear urine stains.
Acknowledgements This work was supported in part Grants-in-Aid
107
from the Japanese Society for the Promotion of Science (12357003 to T.Y. and 12307011 to K.K.).
References [1] Kishi K, Yasuda T, Takeshita H. DNase I: structure, function, and use in medicine and forensic science. Legal Med 2001;3: 69– 83. [2] Nadano D, Yasuda T, Kishi K. Measurement of deoxyribonuclease I activity in human tissues and body fluids by a single radial enzyme-diffusion method. Clin Chem 1993;39:448–52. [3] Yasuda T, Takeshita H, Kishi K. Genetic polymorphisms detectable in human urine: their application to forensic identification. Jpn J Legal Med 1997;51:407 –16. [4] Brinkmann B, Rand S, Bajanowski T. Forensic identification of urine samples. Int J Legal Med 1992;105:59–61. [5] Yasuda T, Iida R, Takeshita H, Ueki M, Nakajima T, Mogi K, Kaneko Y, Tsukahara T, Kishi K. A simple method of DNA extraction and STR typing from urine samples using a commercially available DNA/RNA extraction kit. J Forensic Sci 2003;48:108 –10. [6] Yasuda T, Mizuta K, Ikehara Y, Kishi K. Genetic analysis of human deoxyribonuclease I by immunoblotting and the zymogram method following isoelectric focusing. Anal Biochem 1989;183:84–8. [7] Sawazaki K, Yasuda T, Nadano D, Iida R, Takeshita H, Uchide K, Kishi K. Deoxyribonuclease (DNaseI) typing from semen stains: low enzyme activity in vaginal fluids does not interfere with seminal DNase I typing from mixture stains. J Forensic Sci 1993;38:1051 –62. [8] Yasuda T, Takeshita H, Sawazaki K, Iida R, Kishi K. Successful deoxyribonuclease I (DNase I) phenotyping from a small piece of used sock. Electrophoresis 1997;17:1253–6. [9] Iida R, Sawazaki K, Yasuda T, Tsubota E, Aoyama M, Matsuki T, Kishi K. Deoxyribonuclease I phenotyping from saliva stains. J Forensic Sci 1999;44:175–8. [10] Kishi K, Takizawa H, Yamamoto S. Forensic serology: Illustrated technical manual. Tokyo, Tokyo: Kanehara; 1990. [11] Yasuda T, Takeshita H, Iida R, Kogure S, Kishi K. A new allele, DNASE1*6, of human deoxyribonuclease I polymorphism encodes an Arg to Cys substitution responsible for its instability. Biochem Biophys Res Commun 1999;260:280–3.
Brief communication
Usefulness of deoxyribonuclease I (DNase I) polymorphism for individualization from small aged urine stains Toshihiro Yasudaa, Haruo Takeshitab, Misuzu Uekia, Tamiko Nakajimab, Kouichi Mogib, Yasushi Kanekob, Reiko Iidac, Koichiro Kishib,* a
b
Department of Biology, Fukui Medical University, Fukui 910-1193, Japan Department of Legal Medicine, Gunma University School of Medicine, Gunma 371-8511, Japan c Department of Legal Medicine, Fukui Medical University, Fukui 910-1193, Japan
Received 21 February 2003; received in revised form 18 March 2003; accepted 20 March 2003
Abstract We devised a procedure that combines a simple extraction method, isoelectric focusing and activity staining using the dried agarose film overlay method, for deoxyribonuclease I (DNase I) typing from aged urine stains. DNase I types were determined without difficulty from urine stains kept at room temperature for 3 months or more in all of the samples tested. The amounts of urine stains required for typing after 3 months of storage were estimated to be equivalent to 60 – 120 ml of liquid urine. Therefore, considering that useful PCR-based DNA typing has not yet been developed for urine stains, DNase I polymorphism could be considered the first biochemical marker found to be well suited for individualization from small aged urine stains. q 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Deoxyribonuclease I polymorphism; Isoelectric focusing; Stains; Urine; Forensic individualization
1. Introduction Deoxyribonuclease I (DNase I; E.C.3.1.21.1) activity is distributed widely in human body fluids, and extremely high levels of the activity are detectable in urine [1,2]. Genetic polymorphisms including biochemical markers [3] and DNA markers [4,5] have been identified and employed for the individualization of liquid urine samples. However, most urine samples encountered by practicing forensic biologists in casework situations are dried stains, and genetic markers suitable for personal identification * Corresponding author. Tel.: þ81-27-220-8030; fax: þ 81-27220-8035. E-mail address: [email protected] (K. Kishi).
from such samples are extremely limited: useful PCRbased DNA typing method has not yet been developed for urine stains. Therefore, it is essential to devise a new and effective system of discrimination from aged urine stains. In this communication, we describe a novel method for DNase I phenotyping from small aged urine stains.
2. Materials and methods 2.1. Biological samples Urine samples were collected from healthy Japanese volunteers including laboratory workers and students,
1344-6223/03/$ - see front matter q 2003 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S1344-6223(03)00049-X
106
T. Yasuda et al. / Legal Medicine 5 (2003) 105–107
all of whom had provided written informed consent. The samples were concentrated, dialyzed and lyophilized as a control material according to a method described previously [6]. A 0.05% w/v solution of the lyophilized materials, corresponding to fivefold concentrated urine, was used for DNase I phenotyping. Artificial urine stains of three common DNase I phenotypes (1, 1 – 2 and 2) were prepared on both filter paper (No. 526, Toyo Roshi Kaisha Ltd., Tokyo, Japan) and two kinds of cloth made of cotton and a mixture of cotton and polyester fiber. A small sheet of each material measuring 2.5 £ 2.5 cm, on which 1 ml of urine freshly collected from each individual was dropped and absorbed, was air-dried for 1 day at room temperature, and stored either at room temperature or at 4 8C, until required for DNase I phenotyping. 2.2. Extraction of DNase I from urine stains Extraction of DNase I from urine stains was performed essentially according to the microextraction method reported previously [7,8], except that 0.1 M MES buffer (pH 6.5) containing 10 mM MgCl2 and 0.1 mM CaCl2 was used as the extraction buffer. The extract recovered from each urine-stained sheet was dialyzed against 0.05% w/v glycine and then lyophilized. The resulting materials were dissolved in 50 ml 0.05% w/v BSA and used for subsequent analysis. 2.3. DNase I phenotyping Isoelectric focusing on a thin layer of polyacrylamide gel (IEF-PAGE) for DNase I phenotyping was performed by a method described previously [6]. Samples for phenotyping were treated with an equal volume of 5 unit/ml Clostridium perfringens neuraminidase (type V, Sigma, St. Louis, MO, USA) overnight at 4 8C before electrophoresis. Following the electrophoretic run, visualization of DNase I was achieved using the dried agarose film overlay (DAFO) method [6].
3. Results and discussion In our previous studies on successful DNase I phenotyping from saliva stains [9] and used socks [8], incorporation of a clean-up step using
Phenyl-Sepharose CL-6B column chromatography prior to electrophoresis was indispensable in order to avoid formation of faint and indistinct bands during IEF-PAGE analysis. However, the extraction procedure without such a clean-up step allowed DNase I to be phenotyped from aged urine stains without difficulty, perhaps due to the higher content of DNase I in urine than in other body fluids [2]. Furthermore, although detergent, BSA or urea had been added to the extraction solution in order to increase the efficiency of DNase I extraction from other body fluid stains, even the buffer optimal for DNase I reaction without addition of such chemicals was found to be effective for DNase I extraction from urine stains: the recovery of DNase I activity extracted from each of the urine stains kept at room temperature for 1, 2, 3 and 4 weeks was estimated to be 90 ^ 7%, 75 ^ 19%, 74 ^ 7% and 65 ^ 10%, respectively, compared with that in freshly collected liquid urine. Also, addition of detergents, such as NP-40 suitable for DNase I extraction from saliva stains [9], to the extraction solution had no favorable effect on extraction of DNase I from the aged urine stains. Therefore, the simple extraction procedure without a clean-up step as described in Section 2 was employed for DNase I typing from urine stains throughout this study. All the stains kept at room temperature or 48C for up to three months or more were correctly typed without false interpretation. The amount of urine stain required for DNase I phenotyping was estimated to be a 0.4– 0.8 cm2 spot on filter paper (equivalent to 60 –120 ml of liquid urine) after 3 months of storage, irrespective of the storage temperature. These amounts of urine are sufficient to perform DNase I phenotyping from items of evidence submitted in criminal investigations. Furthermore, a longer incubation with the DAFO sheet or a slight elevation of the incubation temperature permits DNase I to be typed from much smaller and older aged urine stains. Since urine samples recovered from crime scenes are commonly encountered as stains dried on cloth, the effect of materials on which liquid urine is absorbed on DNase I phenotyping from these stains should be examined. Phenotyping from two kinds of urine-stained cloth made of cotton and a mixture of cotton and polyester fiber could be performed in the same manner as for urine-stained filter paper. Therefore, it could be concluded that success or failure of
T. Yasuda et al. / Legal Medicine 5 (2003) 105–107
DNase I phenotyping from urine stains does not depend on the material components of the cloth on which the urine has been deposited. Few genetic markers are applicable for personal identification from dried urine stains, which are commonly found at crime scenes; only ABO phenotyping is used in practice, and is useful only if the stains have been deposited by secretor-type persons [10]. Since high levels of DNase I activity were found to be conserved on aged urine stains, urinary DNA may be too badly degraded during storage of the stains to be typed. In fact, correct STR typing could not be performed from a liquid urine samples kept for only 1 week at room temperature [5]. Furthermore, the low content and relatively labile nature of the genetic biochemical markers characterized in urine have previously hindered the application of these markers to individualization of urine stains. On the other hand, the favorable distribution of the DNase I gene frequencies [1], the highly stable nature of the DNase I protein [11] and the high levels of its activity in human urine allow DNase I polymorphism to be well suited for this purpose. Availability of a highly sensitive phenotyping method employing DAFO activity staining has enabled DNase I typing to be performed on aged body fluid stains including urine stains. Therefore, it is concluded that DNase I polymorphism is the first genetic marker to be found effective for forensic individualization of urine stains. Biochemical markers like DNase I polymorphism, which can be detected by conventional, but well approved and established methods, can be useful for forensic investigations of criminal materials if they bear urine stains.
Acknowledgements This work was supported in part Grants-in-Aid
107
from the Japanese Society for the Promotion of Science (12357003 to T.Y. and 12307011 to K.K.).
References [1] Kishi K, Yasuda T, Takeshita H. DNase I: structure, function, and use in medicine and forensic science. Legal Med 2001;3: 69– 83. [2] Nadano D, Yasuda T, Kishi K. Measurement of deoxyribonuclease I activity in human tissues and body fluids by a single radial enzyme-diffusion method. Clin Chem 1993;39:448–52. [3] Yasuda T, Takeshita H, Kishi K. Genetic polymorphisms detectable in human urine: their application to forensic identification. Jpn J Legal Med 1997;51:407 –16. [4] Brinkmann B, Rand S, Bajanowski T. Forensic identification of urine samples. Int J Legal Med 1992;105:59–61. [5] Yasuda T, Iida R, Takeshita H, Ueki M, Nakajima T, Mogi K, Kaneko Y, Tsukahara T, Kishi K. A simple method of DNA extraction and STR typing from urine samples using a commercially available DNA/RNA extraction kit. J Forensic Sci 2003;48:108 –10. [6] Yasuda T, Mizuta K, Ikehara Y, Kishi K. Genetic analysis of human deoxyribonuclease I by immunoblotting and the zymogram method following isoelectric focusing. Anal Biochem 1989;183:84–8. [7] Sawazaki K, Yasuda T, Nadano D, Iida R, Takeshita H, Uchide K, Kishi K. Deoxyribonuclease (DNaseI) typing from semen stains: low enzyme activity in vaginal fluids does not interfere with seminal DNase I typing from mixture stains. J Forensic Sci 1993;38:1051 –62. [8] Yasuda T, Takeshita H, Sawazaki K, Iida R, Kishi K. Successful deoxyribonuclease I (DNase I) phenotyping from a small piece of used sock. Electrophoresis 1997;17:1253–6. [9] Iida R, Sawazaki K, Yasuda T, Tsubota E, Aoyama M, Matsuki T, Kishi K. Deoxyribonuclease I phenotyping from saliva stains. J Forensic Sci 1999;44:175–8. [10] Kishi K, Takizawa H, Yamamoto S. Forensic serology: Illustrated technical manual. Tokyo, Tokyo: Kanehara; 1990. [11] Yasuda T, Takeshita H, Iida R, Kogure S, Kishi K. A new allele, DNASE1*6, of human deoxyribonuclease I polymorphism encodes an Arg to Cys substitution responsible for its instability. Biochem Biophys Res Commun 1999;260:280–3.