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1.
Int J Legal Med ; 116(3): 191-4, 2002 Jun.
Article in English | MEDLINE | ID: mdl-12111327

ABSTRACT

From sequence database information we have newly identified three male-specific and polymorphic tetranucleotide STRs, DYS443 (GDB: 10807127), DYS444 (GDB: 10807128) and DYS445 (GDB: 10807129) on the Y chromosome. Analysis of 190 Japanese males revealed 6, 5 and 4 alleles in the DYS443, DYS444 and DYS445 systems, with calculated STR diversities of 0.68, 0.57 and 0.53, respectively. The cumulative haplotype diversity of the five Y-STRs DYS441, DYS442, DYS443, DYS444 and DYS445 was calculated to be 0.95 and therefore application of these STRs may yield very useful information for forensic individualization.


Subject(s)
Haplotypes , Polymorphism, Genetic , Tandem Repeat Sequences/genetics , Y Chromosome , Base Sequence , DNA/genetics , Forensic Medicine/methods , Humans , Japan , Male , Molecular Sequence Data
2.
J Forensic Sci ; 44(1): 175-8, 1999 Jan.
Article in English | MEDLINE | ID: mdl-9987884

ABSTRACT

Good typing results were obtained using a newly developed method for extraction and purification of deoxyribonuclease I (DNase I) from saliva stains. Previously, DNase I phenotyping from saliva stains has been unsuccessful because of low enzyme activity and heavy contamination. Salivary DNase I was extracted from stains using phosphate buffer containing Nonidet P-40. Extracts were purified using Phenyl Sepharose CL-4B gel. Electrophoresis was performed, and DNase I was successfully phenotyped. All of the DNase I phenotypes, which were obtained from saliva stains using this new method, were identical to the phenotypes determined from urine samples. Moreover, DNase I was correctly phenotyped from saliva stains that had been stored for over three months at room temperature or at 37 degrees C. These results suggest that DNase I polymorphisms provide valuable information for forensic characterization of saliva stains.


Subject(s)
Deoxyribonuclease I/analysis , Saliva/enzymology , Coloring Agents , Humans , Hydrogen-Ion Concentration , Isoelectric Focusing , Phenotype
3.
Electrophoresis ; 19(14): 2416-8, 1998 Oct.
Article in English | MEDLINE | ID: mdl-9820960

ABSTRACT

A highly sensitive method for detecting deoxyribonucleases (DNases) I and II on an electrophoresed gel is described. A dried agarose film sheet containing DNA as a substrate and a buffer reagent was placed in contact with the gel surface after electrophoresis (DAFO method, Yasuda et al., Anal. Biochem. 1989, 183, 84-88). After an appropriate incubation period, the film sheet was peeled off and stained with SYBR-Green I (SG), and then the DNase isozyme bands were detected using a fluorescence image analyzer. We could detect pg levels of the DNases (DNase I, 2 pg; DNase II, 2pg), which represents a 32- to 128-fold increase in sensitivity compared with the original DAFO method using ethidium bromide (EB) as the fluorescent dye. A combination of this new detection method and isoelectric focusing electrophoresis in polyacrylamide gel allowed accurate DNase I typing from 1 microL human serum. This new technique has been named SG-DAFO, after its original dried agarose film overlay method using EB (EB-DAFO).


Subject(s)
Deoxyribonuclease I/blood , Electrophoresis, Agar Gel/methods , Electrophoresis, Polyacrylamide Gel/methods , Endodeoxyribonucleases/blood , Fluorescent Dyes , Isoenzymes/analysis , Organic Chemicals , Benzothiazoles , Diamines , Ethidium , Humans , Isoelectric Focusing , Microchemistry , Quinolines , Sensitivity and Specificity
4.
J Forensic Sci ; 41(5): 862-4, 1996 Sep.
Article in English | MEDLINE | ID: mdl-8789848

ABSTRACT

We confirmed for the first time, both biochemically and immunologically, the existence of deoxyribonuclease I (DNase I) in human liquid sweat. Isoelectric focusing of sweat samples on polyacrylamide gels (pH 3.5 to 5), followed by dried agarose film overlay detection, was used to determine the phenotypes of sweat DNase I. Because this detection method not only had high sensitivity, but also high band resolution, it was possible to determine DNase I types from sweat samples of 50 to 100 microL. Pretreatment of sweat samples with sialidase was essential for typing to enhance markedly the sensitivity accompanied by simplification of the isozyme pattern. The DNase I types in all sweat samples were consistently related to the types found in corresponding blood, urine, and semen samples. DNase I typing could, therefore, provide a novel discriminant characteristic in the forensic examination of sweat.


Subject(s)
Deoxyribonuclease I/analysis , Sweat/enzymology , Biomarkers/analysis , Biomarkers/blood , Biomarkers/urine , Deoxyribonuclease I/blood , Deoxyribonuclease I/urine , Humans , Male , Phenotype , Polymorphism, Genetic , Semen/enzymology
5.
Electrophoresis ; 17(7): 1253-6, 1996 Jul.
Article in English | MEDLINE | ID: mdl-8855413

ABSTRACT

The forensic application of the DNase I polymorphism for individualization of used socks is described. We devised a technique that combines a simple extraction method, including a partial purification step, using a Phenyl-Sepharose CL-4B column, isoelectric focusing and activity staining, for DNase I typing from socks. As a result, DNase I types were determined from socks kept at room temperature for a month or more in all of the samples tested. The DNase I types determined from socks exactly matched those determined from corresponding sweat and urine samples. By contrast, AB0 groupings could not be determined from these samples, using the conventional serological method. Therefore, we have shown that the DNase I polymorphism is the first genetic marker found to be well suited for individualization of used socks.


Subject(s)
Clothing , Deoxyribonuclease I/genetics , Deoxyribonuclease I/isolation & purification , Forensic Medicine , Genetic Markers , Phenotype , ABO Blood-Group System , Blood Grouping and Crossmatching , Electrophoresis, Polyacrylamide Gel , Humans , Isoelectric Focusing
6.
Electrophoresis ; 17(4): 793-6, 1996 Apr.
Article in English | MEDLINE | ID: mdl-8738346

ABSTRACT

Polymorphism of alpha-2-HS-glycoprotein (AHSG) was demonstrated in human semen and whole saliva samples by thin-layer polyacrylamide gel isoelectric focusing (IEF) and immunoblotting. Although the seminal AHSG IEF patterns were found to differ from those of plasma AHSG from the corresponding donors, incorporation of Nonidet P-40 into the IEF gel (pH 4.2-4.9) enabled us to phenotype seminal AHSG correctly. Salivary AHSG, however, exhibited IEF patterns similar to those of the corresponding plasma AHSG. By treating the samples with neuraminidase, it was possible to determine the AHSG types using 2-5 microL semen and 50-100 microL whole saliva samples. The AHSG types determined separately in 47 sets of semen, whole saliva, urine and plasma samples from the same donors correlated perfectly with each other. AHSG typing could, therefore, provide an additional discriminant characteristic in the forensic examination of semen and saliva samples.


Subject(s)
Blood Proteins/analysis , Saliva/metabolism , Semen/metabolism , Electrophoresis, Polyacrylamide Gel/methods , Humans , Immunoblotting , Isoelectric Focusing/methods , Male , Polymorphism, Genetic , alpha-2-HS-Glycoprotein
7.
Protein Expr Purif ; 7(2): 167-72, 1996 Mar.
Article in English | MEDLINE | ID: mdl-8812855

ABSTRACT

Affinity chromatography on immobilized ribonuclease (RNase) inhibitor was developed for purification of mammalian RNase. Human placental RNase inhibitor was conjugated to CNBr-activated Sepharose in the presence of dithiothreitol. About 80% of the immobilized RNase inhibitor was capable of binding bovine pancreatic RNase A. The bound RNase A was eluted with 3 M NaCl at pH 5.0. Two 25-kDa and 18-kDa RNases, which were obtained from human liver using a cellulose phosphate column, were bound to the immobilized RNase inhibitor and recovered in a pure and active form after treatment of the resin with p-hydroxymercuribenzoate. These enzymes were considered to be nonsecretory-type RNases with different sugar contents.


Subject(s)
Liver/enzymology , Placental Hormones/metabolism , Ribonucleases/isolation & purification , Amidohydrolases/metabolism , Animals , Cation Exchange Resins/metabolism , Cattle , Cellulose/analogs & derivatives , Cellulose/metabolism , Chromatography, Affinity/methods , Electrophoresis, Polyacrylamide Gel , Female , Humans , Hydrogen-Ion Concentration , Isoenzymes/isolation & purification , Molecular Weight , Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase , Placenta/enzymology , RNA/metabolism , Ribonucleases/antagonists & inhibitors , Ribonucleases/metabolism , Substrate Specificity
8.
Electrophoresis ; 17(1): 104-9, 1996 Jan.
Article in English | MEDLINE | ID: mdl-8907526

ABSTRACT

A simple method for the separation and specific detection of basic ribonucleases (RNases) was developed. The separation was achieved by polyacrylamide gel electrophoresis in a pH gradient generated by a carrier ampholyte (Pharmalyte 8-10.5) and arginine. In order to prevent interference from atmospheric carbon dioxide, the pH gradient was formed in sealed vertical gel slab. Human nonsecretory-type RNase, bovine pancreatic RNase A, and other basic proteins could be resolved without expensive equipment or complicated procedures. For activity detection after electrophoresis a zymogram technique was applied, using dry agarose film containing ethidium bromide plus RNA as substrate. This approach affords two advantages: (i) Basic RNase activities can be detected within 15 min, even in crude materials. The sensitivity is better than 0.5 ng of purified human nonsecretory-type RNase. (ii) An inhibition test of RNase activities in the gel, using human placental-type RNase inhibitor, can be performed.


Subject(s)
Alkalies/analysis , Electrophoresis, Polyacrylamide Gel/methods , Ribonucleases/analysis , Buffers , Catalysis , Enzyme Inhibitors/pharmacology , Humans , Hydrogen-Ion Concentration , Reference Values , Ribonuclease, Pancreatic/analysis , Ribonuclease, Pancreatic/antagonists & inhibitors , Ribonucleases/antagonists & inhibitors , Sensitivity and Specificity
9.
Electrophoresis ; 16(10): 1889-93, 1995 Oct.
Article in English | MEDLINE | ID: mdl-8586059

ABSTRACT

We recently completely elucidated the molecular basis of genetic polymorphism in human deoxyribonuclease I and found it to be controlled by four codominant alleles, DNASE1*1, *2, *3 and *4. In this paper we describe a novel DNase I-genotyping system that could be used directly on DNA samples using the polymerase chain reaction (PCR) based on the three nucleotide substitutions underlying the protein polymorphism. The system consists of three independent reactions. Since the substitutions neither suppress nor create any known enzyme recognition site in the DNase I gene, two separate mismatched PCR followed by XhoI digestion methods were introduced to discriminate between the DNASE1*1 (or *3) and the DNASE1*2 (or *4) alleles, and to detect the DNASE1*4 allele. An amplification refractory mutation system was employed to detect DNASE1*3. A 100% correlation was found between the results of this genotyping method and those obtained by phenotyping using conventional isoelectric focusing. The high sensitivity and specificity of this genotyping method allows us to survey DNase I-polymorphism in small DNA samples.


Subject(s)
Deoxyribonuclease I/genetics , Genotype , Polymerase Chain Reaction , Polymorphism, Restriction Fragment Length , Alleles , Base Sequence , Deoxyribonucleases, Type II Site-Specific/metabolism , Exons , Humans , Molecular Sequence Data
10.
Nihon Hoigaku Zasshi ; 49(2): 65-9, 1995 Apr.
Article in English | MEDLINE | ID: mdl-7783389

ABSTRACT

The correlation between isoprotein types of alpha-1-antitrypsin (PI) in blood and semen samples from the same individual was determined in 48 Japanese men by the combined technique of isoelectric focusing with immobilized pH gradients and immunoblotting. Five common PI types (M1, M1M2, M1M3, M2 and M2M3) were detected in the blood plasma samples. However, PI-specific bands in semen migrated more cathodally than those in plasma into a pI region of approximately 5.05, and about 17% of the semen samples could not be phenotyped: the rest were easily phenotyped and their PI types were found to correlate with the type found in the corresponding blood samples. PI typing could therefore provide an additional discriminant characteristic for the forensic examination of individualization from semen samples.


Subject(s)
Forensic Medicine , Polymorphism, Genetic , Semen/enzymology , alpha 1-Antitrypsin/genetics , Humans , Hydrogen-Ion Concentration , Immunoblotting , Isoelectric Focusing , Male , alpha 1-Antitrypsin/classification
11.
FEBS Lett ; 359(2-3): 211-4, 1995 Feb 13.
Article in English | MEDLINE | ID: mdl-7867802

ABSTRACT

In addition to the three alleles commonly responsible for the protein polymorphism of human deoxyribonuclease I, a mutation encoded by a fourth allele, DNASE1*4, was detected by isoelectric focusing. All 8 exons covering the entire open reading frame of the human DNase I gene were amplified by the polymerase chain reaction and subjected to direct sequencing. Only one nucleotide substitution, a C-to-G transition (CAG-->GAG), in the codon for amino acid 9 of the mature enzyme was found. This substitution resulted in the replacement of Gln with Glu (Q9E).


Subject(s)
Alleles , Deoxyribonuclease I/genetics , Polymorphism, Genetic , Amino Acid Sequence , Animals , Base Sequence , DNA Mutational Analysis , DNA Primers , Humans , Molecular Sequence Data , Mutation , Polymerase Chain Reaction , Sequence Homology, Amino Acid
12.
Hum Genet ; 94(3): 224-30, 1994 Sep.
Article in English | MEDLINE | ID: mdl-8076935

ABSTRACT

The main isozyme patterns of desialylated blood plasma or serum alpha-L-fucosidase (FUCA) were found to be almost identical to those of semen, urine, placental extracts, and leukocyte lysates, when detected by polyacrylamide gel isoelectric focusing, and activity staining using the fluorogenic substrate 4-methylumbelliferyl-alpha-L-fucopyranoside. Three phenotypes (1, 2-1, and 2) determined from plasma samples were identical to the phenotypes from urine and leukocyte lysates from the same individuals. A population study of plasma samples collected from 485 Japanese individuals indicated that the frequencies of the FUCA1*1 and FUCA1*2 alleles were 0.7505 and 0.2495, respectively. The mean plasma enzyme activities (+/- SD) of the three phenotypes were 318.8 +/- 116.7 nmol/ml per h for type 1, 268.0 +/- 108.3 nmol/ml per h for type 2-1, and 233.2 +/- 84.4 nmol/ml per h for type 2. The mean activities of types 1 and 2 suggest that, on average, the FUCA1*1 gene product in plasma has about 1.4 times the activity of FUCA1*2.


Subject(s)
Isoenzymes/genetics , Polymorphism, Genetic , alpha-L-Fucosidase/genetics , Adult , Alleles , Child , Electrophoresis, Polyacrylamide Gel , Female , Gene Frequency , Humans , Isoelectric Focusing , Isoenzymes/blood , Isoenzymes/urine , Leukocytes/enzymology , Male , Middle Aged , Phenotype , Placenta/enzymology , Pregnancy , Semen/enzymology , alpha-L-Fucosidase/blood , alpha-L-Fucosidase/urine
13.
Int J Biochem ; 26(8): 1025-31, 1994 Aug.
Article in English | MEDLINE | ID: mdl-8088412

ABSTRACT

Deoxyribonucleases (DNases) I and II activities in 13 different organs and body fluids from healthy male rabbits were measured using the single radial enzyme diffusion method. We now show that testis, epididymis, ampulla, seminal vesicle, vesicular gland, prostate, and semen have both of the DNases I and II activities, whereas spermatozoa do not. DNase I activities were highest in epididymis and seminal vesicle, whereas DNase II activities were highest in epididymis and prostate among the reproductive organs. The presence of these two enzyme activities outside the digestive system suggests that they have another biological function in addition to their digestive roles.


Subject(s)
Deoxyribonuclease I/metabolism , Endodeoxyribonucleases/metabolism , Genitalia, Male/enzymology , Animals , Hydrogen-Ion Concentration , Isoelectric Focusing , Male , Rabbits , Semen/enzymology , Spermatozoa/enzymology , Testis/enzymology
14.
Electrophoresis ; 14(10): 1042-4, 1993 Oct.
Article in English | MEDLINE | ID: mdl-8125053

ABSTRACT

Isoelectric focusing of whole saliva samples on polyacrylamide gels (pH 3.5-5), followed by dried agarose film overlay detection, was employed to determine the type of salivary deoxyribonuclease I (DNase I). Since this detection method had not only a high sensitivity, but also a high band resolution, it was possible to determine DNase I types from saliva samples of 2-5 microL. Pretreatment of saliva samples with neuraminidase simplified the isozyme pattern and enhanced the sensitivity. The DNase I types in all 30 saliva samples showed a good correlation with the types found in the corresponding blood, semen, and urine samples. This preliminary trial indicates that DNase I typing from saliva samples is a new and promising method for individualization of casework samples in the field of forensic biology.


Subject(s)
Deoxyribonuclease I/analysis , Deoxyribonuclease I/genetics , Polymorphism, Genetic , Saliva/enzymology , Female , Humans , Isoelectric Focusing/methods , Male , Semen/enzymology , Sensitivity and Specificity , Sepharose
15.
J Forensic Sci ; 38(5): 1051-62, 1993 Sep.
Article in English | MEDLINE | ID: mdl-8228876

ABSTRACT

We describe the use of deoxyribonuclease I (DNase I) polymorphism for individualization of semen in body fluid stain mixtures, as a means of providing new and more useful information to practicing forensic biologists as a genetic marker. We have already reported that human DNase I isozyme patterns from different subjects are classifiable into ten groups. Isoelectric focusing of DNase I isozymes on polyacrylamide gel (IEF-PAGE, pH 3.5 to 5) was accomplished using a 0.5 mm thick gel. Pretreatment of semen samples with neuraminidase enhanced the isozyme band resolution and sensitivity. Activity detection using the dried agarose film overlay (DAFO) procedure was reliable, sensitive and simple, with high resolution, and the phenotypes of DNase I were determined in semen stains of about 0.3 microL stored at room temperature for up to a year in most of the samples tested. The DNase I types in semen stains were correlated with the types found in the corresponding blood and urine samples, although most of the vaginal fluid samples had no typable DNase I activity. This is considerably advantageous for seminal individualization from body fluid mixture stains in criminal cases. An evaluation of DNase I typing by IEF-PAGE and DAFO was also performed on casework samples submitted to our laboratory, and the results showed that DNase I was expected to be one of the most useful individualization marker of semen in practical application.


Subject(s)
Body Fluids/enzymology , Deoxyribonuclease I/analysis , Semen/enzymology , Vagina/enzymology , Deoxyribonuclease I/genetics , Female , Forensic Medicine , Humans , Isoelectric Focusing , Isoenzymes/analysis , Isoenzymes/genetics , Male , Phenotype , Polymorphism, Genetic
16.
Clin Chim Acta ; 218(1): 5-16, 1993 Sep 17.
Article in English | MEDLINE | ID: mdl-8299220

ABSTRACT

Deoxyribonuclease I (DNase I) was purified from the semen of a 38-year-old male and then characterized. The catalytic properties of the purified enzyme closely resembled those of DNase I purified from the urine of this individual and the following other similarities were observed: molecular masses, iodoacetic acid inactivation kinetics, desialylated isoenzyme patterns. However, the behavior of the purified enzymes determined on several different lectin-affinity chromatography columns differed, which suggests that organ-specific glycosylation of DNase I occurs. Multiple forms of the purified seminal DNase I were demonstrated, each of which had a different pI value separated by isoelectric focusing, which is compatible with the reported existence of genetic polymorphism of seminal DNase I (Sawazaki et al., Forensic Sci Int 1992;57:39-44). Furthermore, enzymological and immunological comparisons of purified seminal and urinary and partially purified prostatic DNases I indicated that the prostate may be one of seminal enzyme source tissues.


Subject(s)
Deoxyribonuclease I/isolation & purification , Semen/enzymology , Adult , Cations, Divalent , Deoxyribonuclease I/antagonists & inhibitors , Deoxyribonuclease I/metabolism , Electrophoresis, Polyacrylamide Gel , Humans , Hydrogen-Ion Concentration , Iodoacetates/pharmacology , Iodoacetic Acid , Isoelectric Focusing , Lectins/metabolism , Male , Phenotype , Prostate/enzymology
17.
Anal Biochem ; 212(1): 111-6, 1993 Jul.
Article in English | MEDLINE | ID: mdl-8368482

ABSTRACT

Two analytical methods for human secretory-type ribonuclease, which are based on polycytidylic acid/ethidium bromide fluorescence, have been developed. The first is a method for measurement of secretory-type ribonuclease activity utilizing the radial diffusion of ribonuclease in a thin agarose gel plate containing polycytidylic acid and ethidium bromide. Ribonuclease activity was visualized as a dark circle on a fluorescent background under ultraviolet light after immersing the gel in a cooled acidic solution. The radius of the dark circle was proportional to the amount of the enzyme. This method allows quantitation of human secretory-type ribonuclease down to at least 5 x 10(-5) unit, which corresponds to 60 pg. Secretory-type ribonuclease activity in 18 different human tissues and body fluids was measured. The second method is a zymogram technique for detection of secretory-type ribonuclease after isoelectric focusing, which includes placing a dried agarose film containing polycytidylic acid and ethidium bromide on the focused gel. Human secretory-type ribonuclease (less than 3 x 10(-4) unit) was detected with a high band resolution on the same principle as that of the activity assay described above.


Subject(s)
Ribonucleases/analysis , Body Fluids/enzymology , Diffusion , Electrophoresis, Polyacrylamide Gel , Ethidium , Evaluation Studies as Topic , Female , Humans , Isoelectric Focusing , Male , Poly C , Pregnancy , Ribonucleases/metabolism , Sensitivity and Specificity , Spectrometry, Fluorescence , Tissue Distribution
18.
Int J Legal Med ; 105(4): 239-41, 1993.
Article in English | MEDLINE | ID: mdl-8431404

ABSTRACT

High-performance liquid chromatography was employed to determine the concentration of thiopental in body fluids and tissues in an individual who had died due to intravenous injection of the clinical dose. The blood concentration of thiopental was 0.6 mg/l. Among the 10 tissues examined, the brain and thymus showed the highest level of the drug; 11.9 mg/kg and 7.66 mg/kg, respectively. The results are discussed in the light of the relevant literature.


Subject(s)
Anesthesia, General , Death, Sudden/pathology , Gastroscopy , Thiopental/pharmacokinetics , Adult , Dose-Response Relationship, Drug , Female , Humans , Thiopental/administration & dosage , Tissue Distribution
19.
Forensic Sci Int ; 57(1): 39-44, 1992 Nov.
Article in English | MEDLINE | ID: mdl-1468730

ABSTRACT

We describe a method for obtaining specific and reproducible deoxyribonuclease I (DNase I) typing from liquid semen. Isoelectric focusing of the enzymes on polyacrylamide gel (IEF-PAGE, pH 3.5-5) was accomplished using a 0.5-mm thick gel. The separated isozymes were visualized by a new activity staining method, dried agarose film-overlay (DAFO). Pretreatment of semen samples with neuraminidase markedly enhanced the isozyme-band resolution and sensitivity. The method was simple and reliable, with high resolution and sensitivity. The DNase I types in semen samples were correlated with the types found in corresponding blood and urine samples. DNase I typing could therefore provide an additional discriminant characteristic in the forensic examination of semen.


Subject(s)
Deoxyribonuclease I/analysis , Forensic Medicine/methods , Polymorphism, Genetic , Semen/chemistry , Genetic Markers , Humans , Isoelectric Focusing , Male , Reproducibility of Results
20.
J Forensic Sci ; 37(6): 1514-24, 1992 Nov.
Article in English | MEDLINE | ID: mdl-1453165

ABSTRACT

We describe a method for obtaining nondistorted and reproducible transferrin (TF) typing from liquid semen and semen stains. Isoelectric focusing of TF isoproteins on polyacrylamide gel (IEF-PAGE, pH 4 to 6.5) was accomplished using a 0.5 mm thick gel. The separated isoproteins were then visualized by immunoblotting with TF-specific antibody. Pretreatment of semen samples with neuraminidase enhanced the TF band resolution. The method was reliable, sensitive and simple, with a high resolution. When maintained at room temperature, laboratory-prepared semen stains were TF-typable for up to at least 50 weeks. The TF types in semen stains were correlated with the types found in the corresponding blood and urine samples. TF typing could therefore provide an additional discriminant characteristic in the forensic examination of semen stains. An evaluation of TF typing by IEF-PAGE and immunoblotting was also performed on casework samples submitted to our laboratory.


Subject(s)
Body Fluids/chemistry , Forensic Medicine/methods , Semen/chemistry , Transferrin/analysis , Adult , Female , Humans , Immunoblotting , Isoelectric Focusing , Male , Reference Values , Reproducibility of Results , Vaginal Smears
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