ABO genotyping by polymerase chain reaction.

ABO blood group system's genotyping by polymerase chain reaction in genomic DNA level is developed. The positions of nucleotide 258 and 700 of cDNA from A transferase were used to distinguish A, B, and O alleles by restriction enzyme digestion. To identify the 258th nucleotide, a 199- or 200-bp DNA fragment was amplified by PCR and digested with Kpn I. For the 700th nucleotide, a 128-bp PCR amplified fragment was designed and digested with Alu I. By examining the DNA fragment digested patterns, ABO genotypes were easily determined. Results obtained using this method on 20 ABO-known peripheral blood samples showed that this new technique could provide accurate ABO genotype. Biologic forensic samples, such as, blood stains, saliva stains, semen stains, hair, bone tissue, and semen contaminated with vaginal secretion were also successfully typed. This rapid, sensitive and reliable method should be applicable not only in forensic identification but also in medical examination.     

Real-time polymerase chain reaction quantification of canine DNA

The accurate quantification of target DNA is an important step in the short tandem repeat analysis of forensic biological samples. By utilizing quantification data to control the amount of template DNA in the polymerase chain reaction (PCR), forensic scientists can optimize testing and minimize the consumption of limited samples. The ability to identify and quantify target DNA in mixed-species samples is crucial when it may be overwhelmed by nontarget DNA, as in cases of dog attack. We evaluated two quantitative real-time PCR assays for dynamic range, species specificity, and inhibition by humic acid. While both assays proved to be highly sensitive and discriminating, the Melanocortin-1 Receptor (MC1R) gene Taqman assay had the advantages of a shorter run time, greater efficiency, and safer reagents. In its application to forensic casework, the MC1R assay has been advantageous for quantifying dog DNA in a variety of mixed-species samples and facilitating the successful profiling of individual dogs.     

An evaluation of the polymerase chain reaction method for forensic applications

This paper describes the use of polymerase chain reaction (PCR) for amplifying small amounts of DNA obtained from samples of interest to the forensic scientist. A region of the HLA DQalpha (DQa) locus was amplified in DNA prepared from the following: hair roots, liquid blood, blood-stains, semen and vaginal swabs (semen free and semen contaminated). A population study was conducted using DNA from 78 unrelated individuals. The observed distribution of HLA DQa alleles varied from that reported for an American population but obeyed the Hardy-Weinberg equilibrium. Interpretation problems associated with the PCR technique are discussed.     

Application of species-specific polymerase chain reaction in the forensic identification of tiger species

Globally, tigers are considered to be endangered, and are listed on Appendix I of CITES. A simple test, using a species-specific primer pair, was developed to identify tiger meat, faeces and dried skin, and provide forensic evidence of illegal wildlife trade. The specific fragment of mitochondrial cytochrome b gene was also successfully amplified from raw DNA products extracted from single tiger hairs. This PCR-based approach opens a new avenue to forensic identification of less-than-optimal samples.     

An analysis of single and multi-copy methods for DNA quantitation by real-time polymerase chain reaction

The goal of this paper was to examine and compare two different commercially available approaches to the determination of the relative quantities of autosomal and Y chromosomal DNA using real-time PCR. One, Quantifiler^® Duo, utilizes a TaqMan^® assay with single copy probes for both autosomal human and Y quantification. The other method, Plexor HY^® utilizes a primer quenching assay with multi-copy probes for its quantification of autosomal human and Y chromosomal DNA. To test these approaches we have utilized the NIST Human DNA Quantitation Standard Reference Material 2372, a set of three different NIST human DNA quantification standards, to examine the precision, accuracy and sensitivity of the real-time PCR assays. We also examined data from both systems utilizing casework samples. The results show that both systems produced linear estimates for DNA quantity over a broad range of input DNA. However we did observe some apparent copy number effects when comparing the three different NIST standards which we attributed to issues with sequence variations in the different standards. Overall, the single copy approach provided better accuracy while the multi-copy approach produced better sensitivity. Thus the choice of which system to use should depend upon the goals of the user.     

Detection of species of graft in xenotransplants using arbitrary primed polymerase chain reaction.

Because of a shortage in the availability of human organs, xenografts have been attempted in humans with cardiac, renal, and hepatic failure, despite limited success. Use of xenografts, however, is regulated under law in various countries. In xenotransplant cases related to violation of transplantation law, determination of species of the source of tissue and organ(s) becomes highly essential. Random amplified polymorphic DNA (RAPD) protocols using six sets of arbitrary short-sequenced primers have been standardized for verifying claims of porcine cardiac and renal grafts in human transplantation cases. Six arbitrary primers used were found to generate unique amplicon patterns at 36 degrees C annealing temperature. Among the selected primers, a single primer set having the sequence 5'- GGTGCGGGAA -3' is found to be the most informative in discerning porcine tissue contamination in humans. The patterns obtained were consistent for a particular genome. The grafted organs in the studied case were analyzed to be of porcine origin.     

Detection of polymorphisms of human DNA after polymerase chain reaction by miniaturized SDS-PAGE.

PCR followed by SDS-PAGE in miniaturized non-denaturing gels permits in some cases the identification of single base pair substitutions in small DNA fragments and therefore, the study of human DNA polymorphisms. The usefulness of the system in forensic science is investigated by typing the HLA-DQA1 locus and the VNTR recognized with the probe pMCT118 (locus D1S80) and it shows to be advantageous over previously published methods for typing the MCT118 system, whereas in HLA-DQA1 typing for forensic casework, both dot-blot with ASO probes and this method could be complementary.     

The effect of luminol on presumptive tests and DNA analysis using the polymerase chain reaction.

This study was designed to test the following factors involved with processing luminol treated bloodstained evidence: 1) The reactivity of other presumptive chemical color tests, phenolphthalin (PT) and tetramethylbenzidine (TMB), following the application of the light emitting luminol presumptive test. 2) The effect of different cleanings of various bloody substrates on the luminol test. 3) The effect of different cleanings of various bloody substrates on the ability to obtain DNA suitable for PCR testing. 4) The ability to extract DNA from luminol treated bloodstained substrates using three extraction techniques. 5) The effect of spraying washed and unwashed bloodstains on various substrates with luminol on the ability to correctly type the DNA using PCR. Our findings indicated that luminol did not adversely effect the PCR testing and did not interfere with the PT and TMB presumptive tests for blood. It was determined that the substrate and the method of cleaning were the major factors affecting DNA yield and the ability to type the bloodstains using PCR based technologies.     

Use of polymerase chain reaction of the HLA-DQ alpha system in forensic trace analysis]

The polymerase chain reaction (PCR) can be used for genetic typing of minute amounts of biological stains. This is achieved by in vitro amplification of a well-defined and genetically polymorphic human genomic DNA sequence. Using the HLA-DQ alpha system, a population study was carried out among 212 unrelated individuals of German origin. The usefulness of this system is discussed by presenting examples of its application in forensic casework, i.e. the analysis of mixed (male/female) body fluids as well as segregation studies on embryonic and paraffin-embedded tissue samples.     

Southwest China Han Population data for nine Y-STR loci by multiplex polymerase chain reaction

POPULATION: One hundred and twenty unrelated Han ethnic individuals from Chengdu, southwest China.     

Sex determination of human blood micro-stains and single hairs using specific DNA amplification with polymerase chain reaction

Amplification of Y chromosome specific DNA in vitro enables a rapid and reliable sex determination of human minute traces such as blood stains and hairs. In presence of male DNA a band of 154 bp is visualized by agarose gel electrophoresis after amplification, this band is lacking in case of female DNA alone. Amplification of a sex independent DNA locus (such as a fragment from the alcohol dehydrogenase gene) generates identical reaction products for both sexes. This shows that the absence of a band is not due to the lack of trace DNA. It is possible to perform this technique with as little as 0.5 microliters of blood or with a single hair.     

The detection of RhD antigen of the rhesus system by the real time polymerase chain reaction

A method for the detection of RhD using a DNA preparation and the newly-developed test-system based on the real time polymerase chain reaction is described. The study including a large variety of specimens has demonstrated the applicability of the novel system to forensic medical examination.     

Sex identification of forensic specimens by polymerase chain reaction (PCR): two alternative methods

Sex identification of forensic samples (bloodstains and decomposed tissue) by polymerase chain reaction (PCR) was investigated. Amplification of a segment of the amelogenin gene using a pair of primers revealed both Y- and X-specific bands at the same time. The gene has counterparts in both the X and Y chromosomes and a small deletion in the former made it possible to distinguish them. Analysis of the X-specific band is the most reliable method for sex identification. THe locus includes a single copy gene so a sample of 250 ng/tube of deoxyribonucleic acid (DNA) is required for identification. Amplification of part of the DYZ1 locus was attempted as an alternative method for analysis of infinitesimal amounts of sample. Even DNA from putrefied tissue could be analyzed by PCR because the locus consists of thousands of copies of repeating units pHY10.     

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.     

Y-chromosomal short tandem repeats haplotyping from vaginal swabs using a chelating resin-based DNA extraction method and a dual-round polymerase chain reaction

Reported are 2 autopsy cases in which Y-chromosomal microsatellite short tandem repeats DYS19, DYS389I and II, DYS390, and DYS393 could be haplotyped with vaginal swabs by using a Chelex 100-based DNA extraction method and dual-round polymerase chain reaction. The extraction of DNA from vaginal swabs by using this method was as efficient or more efficient than using proteinase K and phenol-chloroform extraction or the alkaline lysis methods. Y-chromosomal microsatellite short tandem repeats haplotyping based on the dual-round polymerase chain reaction method provided genotypes from all the loci determined. Although amplification of Y-chromosomal microsatellite short tandem repeats loci is not directly involved in the existence of spermatozoa, it is considerably advantageous for male individualization from body fluid mixture stains in criminal cases.     

The development of a test-system for the quantitative and qualitative evaluation of DNA content in criminalistic objects by the real-time polymerase chain reaction

An original test-system for the preliminary quantitative and qualitative evaluation of isolated DNA is proposed by the polymerase chain reaction in real time (PCR-RT) based on the TaqMan technology. This test-system permits to simultaneously measure the amount of DNA in the sample, identify the genetic gender, and detect PCR inhibitors. The method has been approbated in the practical work of forensic medical experts.     

Special educational needs and the law: Some practical implications

This article focuses on the law relating to special educational needs. It discusses the impact which the growth in the area of parental rights has had on LEA administrative and executive functions. It highlights varying circumstances leading up to the processes of statutory assessment and statementing in which parents and the LEA may find themselves in a position of conflict and tension. This article provides an analysis of how one specific LEA has sought to respond to the burgeoning workload and associated pressures brought about by changes in educational legislation, regulations and case law and the general increase in the number of appeals lodged against it.     

礼法之争的现实思考

春秋到战国的几百年时间里,儒法两家关于礼与法的争论一直在进行着。在这场争辩中,两派此消彼长,相互推动,形成了系统的治国方略,构成了我国古代政治思想宝库中极为重要的组成部分,其中有很多思想至今仍然闪烁着灿烂的光芒,有着现实的利用价值。发掘古代思想中的精华,汲取古人为政的经验教训,作为我国社会主义政治体制改革的借鉴,无疑是十分重要而有意义的。 根据德法争论焦点的不同,可以将这场争辩划分为治政内容上的争论与治政方法上的争论两大方面。治政内容方面的争论主要是围绕应该依据礼来治国,还是应该依据法律来治国而…