Genotyping of the ABO blood group system: analysis of nucleotide position 802 by PCR-RFLP and the distribution of ABO genotypes in a German population

Genotypes of the ABO blood group system were studied by PCR-RFLP analysis of the eight polymorphic nucleotide positions (ups) 261, 467, 526, 646, 703, 796, 802 and 803 of the cDNA from A transferase. In 169 unrelated German individuals, 17 genotypes were found and the calculated allele frequencies of A(Pro), A(Leu), B, O(T), O(A) and O² were 0.2130, 0.0770, 0.0473, 0.4260, 0.2160 and 0.0207, respectively. These frequency data may provide useful additional information for disputed paternity and stain testing. A variant O allele, O², was fout at a polymorphic frequency. As the nucleotide (np 261) of the O² allele is the same as that of A and B alleles, the analysis of at least three nucleotide positions, i.e. ups 261, 526 and 802, is necessary to avoid mistyping of the ABO genotype.     

PCR在ABO疑难血型鉴定中的应用

ABO血型抗原是人类最主要的血型抗原，ABO疑难血型的鉴定正成为安全输血的一个重要课题。随着ABO血型基因的成功克隆，PCR分型技术的应用，使ABO型别分析达到了更精细的水平。本文对ABO疑难血型的鉴定现状、PCR鉴定的原理和方法进行了综述。随着分子生物学理论和实验技术的进一步发展，PCR方法在鉴定ABO疑难血型方面将有更广泛的应用前景。     

Production and characterization of monoclonal antibodies against tissue specific epitopes on ABO blood group substances in saliva

Summary Mouse monoclonal antibodies (P4-2F, P45C) against ABO blood group substances in saliva were produced by immunization with ABO blood group active-glycoprotein after ethanol precipitation from heated saliva. These antibodies bound to saliva, irrespective of the ABO blood group and secretor status. Saliva diluted at least 3.2 x 10 -fold could be detected by ELISA using these antibodies. Tissue and species specificity of the antibodies was tested by ELISA and counterimmunoelectrophoresis and showed that the antibodies were specific for human saliva. By immunoblotting of the deglycosylated ABO blood group substances it was evident that the epitopes for the antibodies were localized on the core protein of blood group substances in saliva. These antibodies could be extremely suitable reagents for the identification of saliva in medico-legal examinations. Furthermore, they may be used as capture antibodies in sandwich methods for ABO blood grouping of saliva from mixtures of body fluids.     

Significance of DNA analysis for determination of ABO blood groups from hair and nail of decomposed human remains: a comparison with phenotyping by the absorption-elution method

Seventy samples from 35 decomposed human remains were investigated for ABO histo-blood group phenotypes and genotypes by the absorption-elution method and PCR-RFLP, respectively. Phenotypes could be determined by the absorption-elution method in all cases except for some failures to detect A and H antigens from scalp hair. Genotyping was also usually successfully performed using nails when intact samples were available. The findings using hairs appeared to depend on the postmortem interval. In this series, an inconsistency between ABO phenotyping and genotyping was observed in two cases, suggesting postmortem antigenic alteration in hair and nail. These findings suggested the usefulness of serial examinations by phenotyping and genotyping for reliable ABO blood grouping of badly decomposed remains.     

A sequence of tests of minute human blood stains for human origin identification and ABO blood grouping

Summary A series of examinations is presented for human origin identification and ABO blood grouping of doubtful minute human blood stains. A blood-stained thread (0.5 cm in length) was first tested to identify human origin by microprecipitation method and then the ABO blood type was determined by both a modified absorption-elution test and a modified mixed agglutination. In the continuous tests, the maximum limits of positive reactions of the microprecipitation method, the modified absorption-elution test, and the modified mixed agglutination were 1:640, 1:160, and 1:2,560 diluted blood, respectively. A and B agglutinogens were more sensitively determined than H agglutinogen. Hemagglutinogens of blood stains on cotton threads were more easily detected than those of polyester ones.     

Development of an indirect competitive ELISA for the detection of ABO blood group antigens

We developed an indirect competitive enzyme-linked immunosorbent assay (ELISA) for the detection of ABO blood group antigens in human samples; in particular for blood stains. ABO blood group antigens conjugated to polyacrylamide were used for immobilized antigen. ABO blood group antigens were extracted from blood stains using a novel method involving pre-incubation with proteinase K (PK), followed by heat treatment. The extracts (analytes) were combined with either anti-A or -B monoclonal antibodies (mAbs), and added directly to the antigen-coated wells. The anti-A and -B mAbs were captured by either ABO blood group antigens present in the analyte or by the immobilized blood group antigens. Peroxidase-conjugated anti-mouse IgM antibody was used to detect anti-A and -B mAbs complexed with immobilized blood group antigens, and a colorimetric reaction using o-phenylenediamine/H₂O₂ used for its measurement. The ELISA developed in this study was able to detect blood group antigens in blood, saliva and blood stains. The detection limit for unknown blood, saliva and blood stain were determined as 1:200, 1:32 and 1:16. Overall the ABO blood grouping ELISA can be used with relative ease for the high throughput screening of biological samples for the detection of ABO blood group antigens.     

Genotyping of ABO blood groups by PCR and RFLP analysis of 5 nucleotide positions

The genotyping of ABO blood groups was performed using the polymerase chain reaction (PCR) method. The 4 DNA fragments containing the nucleotide position 261, 526, 703 and 796 of cDNA from A-transferase were amplified by PCR, and the amplified DNA subjected to restriction fragment length polymorphism (RFLP) analysis. The different nucleotide at position 803 was clearly distinguished by electrophoresis of the PCR products amplified with allele-specific primers. By analyzing the electrophoresis patterns, ABO genotyping was conclusively accomplished. The frequencies of ABO genotypes found in Japanese blood donors with A and B phenotypes were as follows: in the phenotype A group, AA =19.8 % and AO = 80.2%; and in the phenotype B group, BB =12.8% and BO=87.2%.     

Immunohistochemical study on the localization of the epitope defined by a human saliva-specific mouse monoclonal antibody (P4-5C)

Summary A novel mouse monoclonal antibody (P4-5C) has been developed which recognizes the core portion of the protein carrying ABO(H) blood group antigens in human saliva. This proved to be specific for human saliva using immunochemical investigations such as enzymelinked immunosorbent assay, Ouchterlony method and counter-immunoelectrophoresis. By licht and electron microscope studies with immunohistochemical techniques using this human saliva-Specific P4-5C as primary antibody, it was shown that P4-5C reacted specifically and exclusively with mucus from the mucous gland cells of human salivary glands. P4-5C reacted neither with the mucous gland cells of other primates (hamadryas baboon, Japanese monkey and Rhesus monkey) and four mammals (dog, cat, rabbit and mouse) nor with other human tissues. The epitope on the core portion of the ABO(H)-carrying protein was defined by P4-5C and could be discriminated from the epitope of ABO(H) blood group antigens using immunoelectronmicroscopy, although these 2 epitopes were localized relatively close to each other. The P4-5C monoclonal antibody can be also used for morphological species identification of tissue specimens from submandibular glands.     

Species identification of blood and bloodstains by high-performance liquid chromatography

Summary A reverse-phase high-performance liquid chromatographic method for species identification of blood and bloodstains is described. The method employs a 300 Å pore SynChropak RP-4 column and ternary solvents (acetonitrile-trifluoroacetic acid-water) and can not only identify a species by its characteristic chromatogram, but also simultaneously demonstrates that it is of blood origin by the existence of the heme peak. Deformations in chromatographic profiles obtained with older bloodstains were observed, but the retention times of heme and the major peaks showed only minor changes. The species could be identified from bloodstains at least 3 months old and the present method has the advantage of simplicity, speed and sensitivity in the practice of forensic science.     

Species identification by means of the cytochrome b gene

Species identification was carried out by nucleotide sequence analysis of the cytochrome b (cytb) gene. The aim of the study was to identify biological specimens from diverse vertebrate animals by extracting and amplifying DNA from 44 different animal species covering the 5 major vertebrate groups (i.e. mammals, birds, reptiles, amphibians and fishes). The sequences derived were used to identify the biological origin of the samples by aligning to cytb gene sequence entries in nucleotide databases using the program BLAST. All sequences were submitted to the GenBank including new species which were not observed in the databases. The applicability of this method to the forensic field is demonstrated by simulated casework conditions where different types of samples including problematic specimens such as hair, bone samples, bristles and feathers were investigated to identify the species. Received: 3 February 1999 / Accepted: 17 December 1999     

Determination of MN blood group from blood stains by electrophoresis and immunoblotting

Summary The determination of blood groups from blood stains is extremely important in medicolegal practice, but there is the possibility of an error in the determination of MN phenotypes by the absorption-elution test. We investigated a new method applying electrophoresis and immunoblotting. As a consequence of various experiments, the most appropriate pretreatment of blood stains was as follows. Blood stains were immersed in physiological saline for 0.5 to 1h and centrifuged. The supernatant was discarded. The sediment was dissolved in sample buffer (TRIS-buffered physiological saline containing 2% sodium dodecyl sulfate) and followed by thermodegradation. It was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After transfer to a nitrocellulose membrane by Western blotting, MN phenotypes could be determined accurately from blood stains by an enzyme immunoassay (EIA) using commercially available polyclonal anti-M and anti-N sera. For blood stains more than 1 month old it was not easy to determine the MN phenotypes.     

Species identification by means of pyrosequencing the mitochondrial 12S rRNA gene

The aim of this study was to develop a new method for species identification based on the analysis of a very short nucleotide sequence. For this reason, the mitochondrial 12S rRNA gene, together with the new method of pyrosequencing, was used. The detection of only 20 nucleotides, following the sequencing primer within a 149-bp fragment by pyrosequencing, was sufficient to identify the biological origin of the samples by alignment with a reference sequence database. A case example with a piece of skin is presented, and the question whether this piece of skin came from a missing wife or from an animal could be answered.     

ABO blood grouping of saliva from mixed body fluids by sandwich methods using monoclonal antibodies to tissue specific epitopes on blood group substance in saliva

Summary In this paper methods for ABO blood grouping of saliva from mixed body fluids have been established. Monoclonal antibodies to tissue specific epitopes on blood group substances in saliva were used as solid phase antibodies to catch the blood group substances. ABO blood grouping of saliva could be performed by these methods without interference from other body fluids (eg. semen, vaginal secretion, urine, sweat and serum). At least 16,000 and 3,000 fold dilutions of secretor saliva were sufficient for ABO blood grouping by sandwich ELISA and sandwich absorption-elution test, respectively.     

Towards simultaneous individual and tissue identification: A proof-of-principle study on parallel sequencing of STRs,amelogenin, and mRNAs with the Ion Torrent PGM

DNA-based individual identification and RNA-based tissue identification represent two commonly-used tools in forensic investigation, aiming to identify crime scene sample donors and helping to provide links between DNA-identified sample donors and criminal acts. Currently however, both analyses are typically performed separately. In this proof-of-principle study, we developed an approach for the simultaneous analysis of forensic STRs, amelogenin, and forensic mRNAs based on parallel targeted DNA/RNA sequencing using the Ion Torrent Personal Genome Machine^® (PGM™) System coupled with the AmpliSeq™ targeted amplification. We demonstrated that 9 autosomal STRs commonly used for individual identification (CSF1PO, D16S539, D3S1358, D5S818, D7S820, D8S1179, TH01, TPOX, and vWA), the AMELX/AMELY system widely applied for sex identification, and 12 mRNA markers previously established for forensic tissue identification (ALAS2 and SPTB for peripheral blood, MMP10 and MMP11 for menstrual blood, HTN3 and STATH for saliva, PRM1 and TGM4 for semen, CYP2B7P1 and MUC4 for vaginal secretion, CCL27 and LCE1C for skin) together with two candidate reference mRNA markers (HPRT1 and SDHA) can all be successfully combined. Unambiguous mRNA-based tissue identification was achieved in all samples from all forensically relevant tissues tested, and STR sequencing analysis of the tissue sample donors was 100% concordant with conventional STR profiling using a commercial kit. Successful STR analysis was obtained from 1 ng of genomic DNA and mRNA analysis from 10 ng total RNA; however, sensitivity limits were not investigated in this proof-of-principle study and are expected to be much lower. Since dried materials with noticeable RNA degradation and small DNA/RNA amplicons with high-coverage sequencing were used, the achieved correct individual and tissue identification demonstrates the suitability of this approach for analyzing degraded materials in future forensic applications. Overall, our study demonstrates the feasibility of simultaneously obtaining multilocus STR, amelogenin, and multilocus mRNA information for combined individual and tissue identification from a small sample of degraded biological material. Moreover, our study marks the first step towards combining many DNA/RNA markers for various forensic purposes to increase the effectiveness of molecular forensic analysis and to allow more forensically relevant information to be obtained from limited forensic material.     

Influence of pairwise genetic distance computation and reference sample size on the reliability of species identification using Cyt b and COI gene fragments in a group of native passerines

Species identification is fundamental to wildlife forensic practice. The desirability of molecular genetic methods is increasing rapidly. The sequence of a marker, rather than its particular diagnostic nucleotides, provides greater safety through comparisons between intra- and inter-specific pairwise genetic distances. However, it has not been well described how reliability of species assignment is influenced by distance computing methods and reference sample sizes. In this study, the influences were tested using 12 species from 4 genera of passerine birds and the sequences of partial Cytochrome b (Cyt b) and Cytochrome Oxidase subunit I (COI) genes. Results showed that different substitution types have different outcomes of pairwise genetic distance estimation and this influences the risk of false inclusion and exclusion. Transition (Ts) is the most effective substitution type to reveal optimal species resolution for both Cyt b and COI gene fragments no matter whether K2P and p-distance are used. Sample size required to accurately estimate pairwise distance is essentially determined by the genetic diversity of a species in reference to a given strictness of predefined acceptable accuracy. These findings suggest that for future forensic work on birds by use of Cyt b and COI gene fragments, transition should be used exclusively for marker validation and identification practice when targeting closely related species. Meanwhile, the reference database should sufficiently represent overall genetic diversity of the species. The minimum sample size should be estimated based on existing knowledge of genetic diversity. Special caution should be used for species assignment when only several reference data are available for animals that are considered likely to have high genetic diversity.     

Applicability of the SNPforID 52-plex panel for human identification and ancestry evaluation in a Brazilian population sample by next-generation sequencing

SNP analysis is of paramount importance in forensic genetics. The development of new technologies in next-generation sequencing allowed processing a large number of markers in various samples simultaneously. Although SNPs are less informative than STRs, they present lower mutation rates and perform better when using degraded samples. Some SNP systems were developed for forensic usage, such as the SNPforID 52-plex, from the SNPforID Consortium, containing 52 bi-allelic SNPs for human identification. In this paper we evaluated the informativeness of this system in a Brazilian population sample (n = 340). DNA libraries were prepared using a customized HaloPlex Target Enrichment System kit (Agilent Technologies, Inc.) and sequenced in the MiSeq Personal Sequencer platform (Illumina Inc.). The methodology presented here allowed the analysis of 51 out of 52 SNPforID markers. Allele frequencies and forensic parameters were estimated, revealing high informativeness: the combined match probability and power of exclusion were 6.48 × 10⁻²¹ and 0.9997, respectively. Population admixture analysis indicates high European contribution (more than 70%) and low Amerindian contribution (less than 10%) in our population, while individual admixture analyses were consistent with the majority of individuals presenting high European contribution. This study demonstrates that the 52-plex kit is suitable for forensic cases in a Brazilian population, presenting results comparable with those obtained using a 16 STR panel.     

Diagnosis of paternity for cases without mother and without both mother and putative father based on blood group findings from the relatives

Summary Formulas of the estimated likelihood ratio Y/X are derived for cases without mother as well as those without both mother and putative father, by using blood group findings of their relatives.The distribution curves of the relative frequencies of log(Y/X) for these cases are calculated with respect to 10⁴ families which are created by a Monte Carlo simulation. The extent of success in the paternity diagnosis is clarified by the statistical analysis based on these distribution curves.According to the above analysis, fairly high chance of success can be obtained in the diagnosis of such ambiguous cases without the plaintive mother and/or the putative father, if their relatives are alive. It is also concluded that the genetic information as to the parents of the deceased person increases the exclusion probability, whereas that as to the spouse and children increases the fraction of log(Y/X) > 1 for non-father, corresponding to the fraction where the Essen-Möller value is less than 9%.     

Descriptive analyses of differentially expressed proteins during intrapuparial stage based on the label-free proteomics technique between Chrysomya megacephala and Synthesiomyia nudiseta

The lack of rapid and accurate species identification methods on pupae restricts the practical application of forensic entomology. It is a new idea to construct portable and rapid identification kits based on the principle of antigen/antibody interaction. Screening differentially expressed proteins (DEPs) of fly pupae is a basis of solving the problem. Here, we used the label-free proteomics technique to discover the DEPs and further validate using the parallel reaction monitoring technique (PRM) in the common flies. In this study, we reared the Chrysomya megacephala and Synthesiomyia nudiseta at constant temperature, and then we sampled at least four pupae at 24 h intervals until the end of the intrapuparial stage. We found 132 DEPs between Ch. megacephala, and S. nudiseta groups, with 68 and 64 proteins being up-regulated and down-regulated between the two groups. Among the 132 DEPs, we selected five proteins having potential for further development and utilization, such as C₁-tetrahydrofolate synthase, Malate dehydrogenase, Transferrin, Protein disulfide-isomerase, and Fructose-bisphosphate aldolase, for further validation using PRM-targeted proteomics, with the trends of PRM results being consistent with the label-free data for corresponding proteins. The present study investigated DEPs via the label-free technique during the pupal development in the Ch. megacephala, and S. nudiseta and provided reference data for development of rapid and accurate identification kits.     

Sex identification on the basis of hand and foot measurements in Indo-Mauritian population--a model based approach

Identification is the foremost issue in crime investigation. A few studies have been performed so far in order to identify sex on the basis of single foot or hand of the victim. Moreover, these studies provide only crude measures to indicate sex and there exists no concrete methodology to predict sex using the available information. In the present paper, we have developed statistical models to identify sex based on the dimensions of foot and hand. The models containing both length and breadth of hand or foot as independent variables are capable of predicting sex in Indo-Mauritian population with fairly high accuracy as compared to those containing hand or foot indices.