The simultaneous electrophoretic analysis of esterase D and phosphoglucomutase subtyping in fresh blood and in dried bloodstains

Phosphoglucomutase1 (PGM) subtyping and esterase D phenotyping were simultaneously performed by electrophoresis of bloodstained fibers using agarose and a Tris-maleic acid buffer system , pH 5.4. This method reduces anodal gel shrinkage and shortens development time when compared to the conventional electrophoretic technique for PGM subtyping which is performed at pH 7.4 using an agarose-starch substrate.     

Simultaneous phenotyping of genetic markers for paternity testing

Time-and cost-saving methods for paternity testing are described. Seventeen genetic systems were divided into six groups: (1) transferrin (Tf), factor B (Bf), and phosphoglucomutase 1 (PGM1); (2) group-specific component (Gc) or alpha 1-antitrypsin (PI) and alpha 2HS-glycoprotein (HSGA); (3) complement components C6 and C7, factor 13B (F13B), and plasminogen (PLG); (4) haptoglobin (Hp), C8 alpha-gamma chain (C81), and factor I (IF); (5) red cell acid phosphatase (ACP), esterase D (ESD), and glutamic-pyruvic transaminase (GPT); and (6) 6-phosphogluconate dehydrogenase (PGD) and glyoxalase I (GLO). Each group of systems was typed simultaneously by electrophoresis or isoelectric focusing (IEF) followed by staining or immunoblotting. These methods are very practical because they afford a considerable saving of time, work and expense, and facilitate semipermanent preservation of electrophoretic patterns.     

The esterase D polymorphism as revealed by isoelectric focusing in ultra-thin polyacrylamide gels

The polymorphism of human red cell esterase D (EsD) was studied using isoelectric focusing (pH 4-6) in ultra-thin polyacrylamide gels. Typing was possible without the EsD isozymes attaining true equilibrium focusing conditions. Using this single method, six phenotypes (EsD 1, 2-1, 2, 5-1, 5-2 and 5) could be recognized in the White population of south-east England. Family studies showed these to be controlled by three co-dominant alleles and the gene frequencies were calculated to be EsD1 0.8856; EsD2 0.0946 and EsD5 0.0198. For successful and reliable EsD typing by this method, the electrophoretic system must be carefully optimized with respect to the duration of electrophoresis and the temperature attained in the gel during the electrophoretic run.     

Population data on the forensic genetic markers: phosphoglucomutase-1, esterase D, erythrocyte acid phosphatase and glyoxylase I

Blood specimens from white and black sample populations from Baltimore, Maryland, were analyzed for the four most forensically important, polymorphic red cell enzyme systems-phosphoglucomutase-1, esterase D, erythrocyte acid phosphatase and glyoxalase I. The distributions of the phenotypes for each marker in each racial group were in Hardy-Weinberg equilibrium. The population data were similar to previously reported data for Whites and Blacks from different geographical locations within the United States.     

The simultaneous separation of the enzymes glyoxalase I, esterase D, and phosphoglucomutase

A procedure for the multisystem analysis of bloodstains using the simultaneous separation of the enzymes glyoxalase I, esterase D, and phosphoglucomutase has been developed. The amount of bloodstain required has therefore been reduced threefold without any loss in resolution and sensitivity. Bloodstains at least seven weeks old have been correctly phenotyped in all three systems.     

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.     

Agarose gel electrophoresis of human erythrocyte glutamic-pyruvic transaminase (GPT EC 2.6.1.2)

The method of choice to determine erythrocyte glutamate-pyruvic transaminase (GPT) including rare variants was starch gel electrophoresis. Methods using agarose as gel medium were not reported to our knowledge. We present an adapted method using the Tris/maleate buffer system and an agarose of low endosmosis. The common as well as rare variant types of GPT were quickly and reliably separated. In addition, a method for the consecutive determination of esterase D (ESD) and GPT on the same gel using the malic acid buffer system is described.     

Mismatched multiplex PCR amplification and subsequent RFLP analysis to simultaneously identify polymorphisms of erythrocytic ESD, GLO1, and GPT genes

ESD (esterase D), GLO1 (glyoxalase I), and GPT (glutamate pyruvate transaminase) are human erythrocytic isoenzymes and have previously been applied in forensic medicine caseworks. The molecular bases of the polymorphic gene expression products have been demonstrated to be because of SNPs in respective coding regions. However, it has not been revealed whether the SNPs conferring the polymorphisms to the aforementioned erythrocytic isoenzymes could be simultaneously detected by using a simple PCR method. In this study, we used mismatched primers to simultaneously amplify three common isoenzyme loci so that all amplified products contained the same Hph I cleavage sites. The products were then digested with Hph I and electrophoretically separated and stained so that alleles were identified. The accumulated values for the probability of discrimination power and excluding the probability of paternity to the aforementioned systems attained 90.41% and 41.72%, respectively, in the Chinese Han population. This assay could be extremely valuable for future forensic medicine practices.     

The effects of heat upon the glyoxalase I isoenzyme

It was observed during the course of routine casework that different bloodstains from the same individual could produce anomalies in the glyoxalase I band patterns. Bloodstains were heated at different temperatures for periods of 4 and 6 h and then examined using electrophoretic techniques. It was demonstrated that upon heating, band alterations in the glyoxalase I Type 1 phenotype can occur, causing the analyst to render the results inconclusive.     

红细胞酯酶D(EsD)型的分布及血痕EsD型的检出

本文应用高压琼脂糖凝胶电泳法对沈阳地区汉族221例随机献血者的红细胞酯酶D(EsD)型进行了检测,其基因频率为 EsD~1=0.629,EsD~2=0.371。结合文献资料分析了EsD 型分布的种族差异,发现黄种人的 EsD~1频率最低,白种人及黑人最高。用本法对红细胞溶血液的最小检出量为1μl;以1μl 溶血液作成的血痕检样,室温保存2周仍可分型。以5μl 溶血液作成的血痕检样可分型时间为3周;37℃保存的溶血液可分型时间为7～8天。     

Esterase D typing of bloodstains by non-equilibrium focusing

Non-equilibrium focusing in a pH 4-6 gradient in ultra-thin polyacrylamide gels has been shown to be a reliable and reproducible method for detecting the six common esterase D phenotypes (EsD 1,2-1,2,5-1,5-2 and 5) in dried bloodstains. Successful typing is dependent on both the age and phenotype of the stain in question. The effects of age on the isozyme pattern of each phenotype are described and illustrated. In a comparative trial using 100 simulated and 300 authentic casework bloodstains, non-equilibrium focusing was shown to be more efficient than thin-layer starch gel electrophoresis for the typing of esterase D.     

用地高辛配基标记的pAW101探针杂交分析DNA多态性及其应用

将NaCl盐析法抽提人基因组DNA与地高辛配基标记DNA探针的方法相结合，检测了15个家系子代与亲代DNApAW101－EcoRI限制性片段的遗传关系。结合所测同工酶（EsD．GLOI．pGM；．Acp）的基因频率、按Essen－moller氏公式计算，父权概率均达到99．73％以上。本法经济、有效、实用、易于在国内普遍实验室开展。     

Applications of isoelectric focusing in forensic serology

The typing of certain polymorphic proteins present in human body fluids is an important aspect of the analysis of serological evidence. This is particularly true when dealing with evidence related to violent criminal activity such as homocide, assault, or rape. Until recently, the routine analysis of the genetic polymorphisms of interest relied upon conventional electrophoretic techniques such as horizontal starch or agarose slab gel or both, cellulose acetate, and vertical polyacrylamide gradient gel methods. These techniques adequately separate a limited number of common variants. In some cases, these methods are still those of choice. However, as a result of the nature of the conventional approach, problems with time required for analysis, resolution, diffusion of bands, sensitivity of protein detection, and cost are often encountered. Isoelectric focusing (IEF) offers an effective alternative to conventional electrophoresis for genetic marker typing. This method exploits the isoelectric point of allelic products rather than charge-to-mass ratio in a particular pH environment. The advantages of employing IEF include: reduction of time of analysis, increased resolution of protein bands, the possibility of subtyping existing phenotypes, increased sensitivity of detection, the counteraction of diffusion effects, and reduced cost per sample.     

Evaluation of a nonequilibrium isoelectric focusing (IEF) method for the simultaneous typing of esterase D (EsD), red cell acid phosphatase (AcP1), phosphoglucomutase (PGM1), adenylate kinase (AK), and adenosine deaminase (ADA)

A nonequilibrium isoelectric focusing method incorporating the chemical spacers MOPS and HEPES was developed and subsequently evaluated for its ability to reliably discriminate common and rare phenotypes in the esterase D (EsD), red cell acid phosphatase (AcP1), phosphoglucomutase (PGM1), adenylate kinase (AK), and adenosine deaminase (ADA) isoenzyme systems. The validation procedures used were blind testing, comparison of results to conventional methods, and evaluation of known rare variant phenotypes. This method proved to be a quick and reliable method for typing all five isoenzyme systems, while providing an excellent probability of discrimination (PD = 0.96).     

A hybrid ampholyte focusing technique for esterase D subtyping of evidentiary material

An ultrathin-layer polyacrylamide gel isoelectric focusing technique that uses a composite of ampholytes from three commercial sources is described for subtyping esterase D. All common allelic products of esterase D were separated clearly. The technique described in this paper provides a higher conclusive call rate on known blood specimens (95.8%) and questioned bloodstains (69.7%) compared with continuous zone electrophoresis in agarose gels (89.9 and 37.6%, respectively).     

Distribution of the AMPFLPs YNZ22, 3′APOB and COL2A1 in the population of Galicia (NW Spain)

Two different electrophoretic methods were used for typing three amplified fragment length polymorphisms (AMPFLPs), (3′ApoB, YNZ22 and COL2A1) in a Galician (NW Spain) population sample. Because of the problems of anomalous mobility for the 3′ApoB system and the intermediate alleles found in the COL2A1 system, the use of automated sequencers and denaturing conditions is recommended for typing these two systems. Nevertheless, simple electrophoretic methods, such as the PhastSystem, can be used for YNZ22 typing. Although intermediate COL2A1 alleles can be distinguished with the sequencers, a binning approach was adopted for comparison purposes. The population sampled was in Hardy-Weinberg equilibrium for the three systems using an exact test. This type of statistical analysis is more appropriate when the number of alleles in a system is high. No significant differences with other Caucasian populations were found for the three systems studied. The characteristics of the polymorphisms, shown by 3′ApoB, YNZ22 and COL2A1, reflected in the statistical parameters studied, demonstrate that these AMPFLPs are of considerable interest for forensic purposes.     

Distributions of genetic markers in United States populations: II. Isoenzyme systems

All published and unpublished population frequency data that could be located for U.S. populations is tabulated and presented for the isoenzyme systems phosphoglucomutase, esterase D, adenylate kinase, acid phosphatase, glyoxalase I, adenosine deaminase, 6-phosphogluconate dehydrogenase, glutamic-pyruvic transaminase, carbonic anhydrase II, and glucose-6-phosphate dehydrogenase. Results obtained by combining data for comparable racial/ethnic groups are also presented. The results obtained with combined data may give better information on frequencies for the U.S. population at large than is obtainable from studies conducted in restricted geographic areas.     

Criminalistics: An introduction to forensic science (2nd edition): By Richard Saferstein. Publisher: Prentice-Hall Inc., Englewood Cliffs,New Jersey,U.S.A., 1981, pp. 470, price $18.95 (cloth)

A technique was developed to type the glyoxalase I (GLO I) isoenzymes using a mixed agarose/starch gel. Over six hundred blood samples from Caucasoid people living in separate regions of South Australia were examined and the results compared with other Caucasoid population surveys. Paired blood and semen samples were also tested and the limitations of the technique with regard to blood and semen stains analysis was evaluated.     

Group specific component: isoelectric focusing subtyping and immunoblot detection

A method for the detection of group specific component (Gc) by immunoblotting, following isoelectric focusing (IEF), is described. This isoelectric focusing method resolves the six common phenotypes of Gc using a narrow range pH 4.5 to 5.4 ampholyte. The Gc proteins were passively transferred from the IEF gel to nitrocellulose and detected with goat anti-Gc followed by peroxidase labeled anti-goat immunoglobulin (Ig) antibody. The increased sensitivity of this technique results in the typing of stains older than one year and also those stains with minimal concentrations of the Gc protein. The polyacrylamide gel can also be used for the subtyping of esterase D.     

EsD*5 gene frequency in Tuscany (Italy)

The distribution of the human red cell esterase D (EsD) "extended" polymorphism in a population sample from Tuscany (Italy) was studied using agarose gel isoelectric focusing. The estimated gene frequencies were: EsD*1 0.864, EsD*2 0.115, EsD*5 0.021. The EsD*5 allele frequency is very similar to those reported for other European populations. The "extension" of the EsD polymorphism may prove to be useful in paternity testing.