An agarose gel electrophoretic method for typing phosphoglucomutase-1, esterase D, or glyoxalase I

A conventional agarose gel electrophoretic method was described for typing phosphoglucomutase-1, esterase D, or glyoxalase I as single systems. Bloodstain extracts were absorbed into 1-mm-thick agarose gels via an application mask. The electrode wick distance was 12 cm and electrophoresis was carried out at 400 V at 6 degrees C. The electrophoretic run times were 30 min for glyoxalase and 1 h for esterase D or phosphoglucomutase. This method is reliable and produces highly resolved band patterns. Additionally, the shorter separation times as a result of the increased voltage gradient permitted typing of more samples in a given time period compared with presently used methods. This technique requires little technical expertise and can be incorporated into the laboratory at a minimal cost.     

Population frequencies of carbonic anhydrase II (CA II), esterase D (EsD), and glyoxalase I (GLO) in the Metropolitan Birmingham, Alabama area

Both black and white populations from Birmingham, Alabama were analyzed for the frequencies of carbonic anhydrase II (CA II), glyoxalase I (GLO) and esterase D (EsD) isoenzymes. The results compared favorably with published frequencies of these genetic markers in other populations.     

Studies on glyoxalase 1 isoenzymes in semen stains: polymorphism in Himachal (India) population

One hundred and ten pairs of blood and semen samples and their stains were studied to type glyoxalase 1 (GLO 1) isoenzymes using agarose-starch medium. A good agreement was observed between the phenotypes expressed in blood and semen samples of the same donor. No GLO 1 activity however could be demonstrated in the vaginal swabs tested. The gene frequencies of GLO 1 polymorphs in Himachal population has been worked out and their stability studies carried out at -12 degrees C and at room temperature.     

DNA typing of forensic material with mixed genotypes using allele-specific enzymatic amplification (polymerase chain reaction).

Biological material in forensic casework frequently contains a mixture of genotypes, with a predominance of material from the victim and only trace amounts from the person committing the crime. Physical separation of the two genotypes or preferential lysis of different cell types may sometimes be possible. However, it is often difficult to achieve complete separation due to the lysis of cells or lack of material. We have developed an enzymatic amplification system for the HLA DQA1 locus, that will allow the presence of individual alleles in a sample with mixed genotypes to be determined, independent of their initial proportion in the sample. This system permits the identification of an allele representing less than 10(-4) of the background genotype. Use of polymerase chain reaction (PCR) with general primers allows only alleles representing more than about 1% to be detected, while the allele-specific amplification represents up to a 1000-fold increase in sensitivity. This method was applied to a rape case and a combined rape and murder case; in both cases the biological evidential materials contained a mixture of alleles from the victim and the rapist. Allele-specific PCR revealed the presence of alleles identical to those of the suspect using DNA from a vaginal swab taken after a rape incident, whereas by using general primers in the PCR only trace amounts of alleles other than those of the victim were found. Similarly, allele-specific amplification of DNA from vaginal swabs from the murder case revealed the presence of alleles identical to those of the suspect, while standard PCR only indicated the presence of genetic material from the victim.     

Lewis typing of human bloodstains by enzyme-linked immunosorbent assay (ELISA) using monoclonal anti-Le(a) and anti-Le(b)

The Lewis blood grouping of human dried bloodstains could be determined by an enzyme-linked immunosorbent assay (ELISA) using monoclonal anti-Le(a) and anti-Le(b) antibodies with an avidin-biotin complex (ABC). The bloodstains aged 1 year were used as samples, and approximately 1 mg of the stains was enough to type each Lewis antigen reliably by this method. The Lewis substances of 106 individual stains were correctly typed regardless of their ABO blood group system.     

Lewis typing of human saliva stains by enzyme-linked immunosorbent assay (ELISA) using monoclonal anti-Le(a) and anti-Le(b) antibodies

The Lewis blood grouping of human saliva stains could be detected by an enzyme-linked immunosorbent assay (ELISA) using anti-Le(a) and anti-Le(b) monoclonal antibodies with an avidin-biotin complex (ABC). The saliva stains (1.0 by 1.0 cm in size) were used as samples and not only could the Lewis substances of 57 individual stains be correctly typed by this method, but also it was clarified that there are several different secretion patterns of amounts of Le(a) and Le(b) substances in 3 individual Lewis types.     

Glyoxalase I typing and phosphoglucomutase-1 subtyping of a single hair

A technique is described for the typing of glyoxalase I (GLO I) and the subtyping of phosphoglucomutase-1 (PGM-1) from the root sheath cells of a single forcibly removed hair. This procedure does not require sample preparation and does not alter the morphological characteristics of the hair. The combined discrimination probability (DP) of the two markers taken together is 0.90 for whites and 0.89 for blacks. GLO I can be typed after four weeks, and PGM-1 can be typed after eight to fifteen weeks in hairs maintained at room temperature. Hairs mounted with Permount showed loss of enzyme activity and loss of band sharpness.     

Haptoglobin typing of human bloodstains using a specific DNA probe

The stability of DNA in human bloodstains and various post mortem tissues has been investigated. High molecular weight (HMW) DNA was usually recovered from dried bloodstains, even those up to a few years old, but very rapid degradation was found to occur post mortem in the liver, pancreas, spleen and kidney. Other tissues such as the heart, thyroid and skeletal muscle were found to give a reasonable yield of HMW DNA during the first few days after death. The feasibility of using DNA extracted from forensic bloodstain specimens for the detection of DNA polymorphisms was explored using a human haptoglobin (Hp) alpha chain specific probe. Using HindIII and XbaI digests the Hp genotypes Hp2, Hp1F and Hp1S were distinguished by Southern blot analysis in DNA prepared from 1 cm2 bloodstains up to 15-18 months old.     

Successful DNA typing of urine stains using a DNA purification kit following dialfiltration

To evaluate the utility of DNA polymorphism typing of urine stains in forensic investigations, the amplifiable amount of DNA was estimated in 20 urine specimens obtained from 10 male and 10 female volunteers using a DNA purification kit following dialfiltration. DNA obtained from both urine and urine stains was amplified with the AmpflSTR Profiler PCR Amplification Kit, and was analyzed by capillary electrophoresis using the Genetic Analyzer. The amount of male and female urine necessary for obtaining a complete DNA profile was 0.2 mL and 0.08 mL, respectively. When 0.2 mL of male urine were used to create urine stains, complete DNA profiles could be obtained from just some of the stains. However, when only 0.1 mL of female urine was used, complete profiles could be successfully obtained from all of the stains. DNA on bleached cotton remained amplifiable for 3-6 weeks. This method using a DNA purification kit following dialfiltration can be recommended for the genotyping of urine stains.     

Direct blood group typing of forensic samples using a simple monoclonal antibody assay

A simple direct test for blood group antigens in samples of blood, dried blood, dried blood associated with fabric, semen, vaginal secretions, saliva and fingerprints is described. This test takes advantage of monoclonal antibodies which have been produced in this laboratory, but which are also becoming available commercially in ever increasing numbers. The test is sensitive and reliable as evidenced by its performance in blind studies of more than 700 blood samples. The test requires no special equipment and can be completed in 4 h. The test is sufficiently versatile that new antibodies can be added to the same test format as they become available.     

法学是一门科学吗?(上)

一、国家立法的实证性与暂时性 在各种科学中,或许没有任何一种和法学一样,人们在表面上似乎可以否定其科学性质,而通过其所经常采用的运作方式,是否也正好[1]可以证立上述判断?[2]让我们先撇开法哲学与法律史不谈,而转向法学当中能够彰显出绝大多数法学家知识与能力之真正领域的那个[3]部分--实证的法学,或者说法教义学--也就是关于在某个国家有效的实证法的学说.     

Report of a European collaborative exercise comparing DNA typing results using a single locus VNTR probe

A collaborative exercise was carried out in 1989 among 12 European forensic laboratories using the single locus VNTR probe pYNH24, the restriction enzyme HinfI, the same set of human genomic DNA samples, and a standardized DNA size marker. The objectives of the exercise were: (1) to study the degree of variation within and between laboratories, (2) to obtain information on requirements for technical standardization allowing the exchange of typing results and (3) to compare different approaches for the identification of allelic DNA fragments of unknown size. Each laboratory carried out up to 10 independent typing experiments using the same DNA samples. The results were analysed independently by two laboratories using three different methods. The results of the exercise demonstrate the correlation of typing that can be achieved within and between laboratories under conditions of minimal standardization.     

A simple method of DNA extraction and STR typing from urine samples using a commercially available DNA/RNA extraction kit

We devised a simple DNA extraction procedure suitable for STR typing of urine sample. Use of a commercially available DNA/RNA extraction kit equipped with a silica-gel-based membrane made it possible to omit the recovery of urinary nucleated cells by sedimentation before the extraction. Successful genotyping of the TH01, HumTPO and multiplex STRs was achieved using aliquots of urine as small as 100 microL. Furthermore, application of this DNA extraction procedure to frozen urine samples provided STR allele results comparable to results obtained from fresh samples. Therefore, this extraction procedure is considered to be effective for STR typing of urine samples in both the frozen and aqueous state. Furthermore, addition of sodium azide to fresh urine samples prolonged their storage duration even at room temperature.     

The typing of group-specific component (Gc protein) in human blood stains

It is known that the typing of group-specific component (Gc protein) in human blood stains is difficult since Gc protein of the extracts of blood stains migrates more anodally to the α₁-globulin region in agar-gel immunoelectrophoresis, while Gc protein in liquid blood normally migrates to the α₂-globulin region. We have reported that the Gc protein found in the α₁-region is the result of binding of actin to Gc protein (Shinomiya, K., Kimura, H., Yoshida, K., and Shinomiya, T., J. Biochem., 92 (1982) 1163–1171, which renders it difficult to determine the Gc-phenotypes in the blood stains. On the basis of the above findings, we developed the method of phenotyping the Gc protein of human blood stains by agar-gel immunoelectrophoresis. Since the binding activity of actin to Gc protein is lost after treatment with a high concentration of guanidine HCl, the extracts of blood stains were treated with 4 M guanidine HCl to dissociate Gc protein and actin and then dialyzed to remove guanidine HCl. By this method we are able to determine the phenotypes of Gc protein in blood stains. The method we have developed is a useful tool in the forensic laboratory.     

Successful DNA typing of a urine sample in a doping control case using human mitochondrial DNA analysis

In a doping control case, a urine sample was tested positive for nandrolon. We were asked by the athlete to perform DNA investigations on the questioned urine sample and compare these to a fresh blood sample taken from the athlete in order to detect or rule out manipulation and/or switching of the samples. The urine sample had been collected nine months prior to the investigation and had been stored at 4 degrees C. In a first approach, nuclear DNA systems were investigated that failed with the exception of the Amelogenin system. Due to the high copy number of mitochondrial DNA molecules and the robustness of the mitochondrial genome, we investigated the HVR I and HVR II regions of mitochondrial DNA and obtained reproducible and clear sequencing results for both the blood and the urine samples. Due to the identical sequences, it could not be excluded that the blood sample and the urine sample were from the same individual or an individual having the same maternal lineage.     

ABO and Lewis typing of secretion stains on nitrocellulose membranes using a new dot-blot-ELISA technique

A new method for ABO and Lewis typing of body fluids is described. It combines the advantages of a good antigen binding to nitrocellulose membranes, the need of only very small amounts of stain material and the high sensitivity of an enzyme-linked immunosorbent assay for antigen detection. This is of special interest because conventional ABO and Lewis typing of secretion stains need relatively large stain dimensions. The method is very easy to handle, does not need any expensive equipment and gives a permanent record. Furthermore the high sensitivity offers the possibility of analyzing even sweat and urine stains without the need of concentrating these extracts.