GC polymorphism detected in human urine by isoelectric focusing and immunoblotting

Genetic polymorphism of GC (vitamin D-binding protein) in human urine was revealed by isoelectric focusing and immunoblotting on thin-layer polyacrylamide gels containing 2 M urea. Urine samples from 530 unrelated Japanese from the Fukui district, being only 1-2 ml of original urine, were examined, and correct GC typing was achieved by comparison with the results of direct grouping using plasma. Six common and twelve rare phenotypes were observed. The frequencies of the genes were 0.473 for GC*1F, 0.241 for GC*1S, 0.254 for GC*2, and 0.032 for the total of six rare alleles.     

Transferrin polymorphism detected in human urine using isoelectric focusing followed by immunoblotting

Genetic polymorphism of transferrin (TF) was revealed in human urine by isoelectric focusing and immunoblotting on thin-layer polyacrylamide gels. Using this technique more than 300 urine samples were examined, and correct TF typing from a small volume of urine (approx. 0.5 ml) was achieved, in comparison with the results of direct grouping for plasma. Three common phenotypes, TF C1, C2-1 and C2, were differentiated. In addition, the rare types TF C1D, C2D, and C1B were observed. The frequencies of the TF alleles in our samples were found to be: TF*C1 = 0.7265, TF*C2 = 0.2624, TF*D = 0.0083 and TF*B = 0.0028.     

The isoelectric focusing of keratins in hair followed by silver staining

An isoelectric focusing method followed by silver staining has been developed for the study of keratins which is as effective as two-dimensional electrophoresis and fluorography for hair species identification. Hair from dogs, rabbits, horses, cows, guinea-pigs, donkeys, sheep and cats were successfully identified. Narrow pH ranges were used to observe heterogeneity in human hair. Although this heterogeneity may be affected by environmental conditions, it may be of use in criminalistics.     

Genetic polymorphism of desialyzed alpha 2 HS-glycoprotein by ultrathin isoelectric focusing

The genetically determined polymorphism of alpha 2 HS-glycoprotein was analyzed by immunoblotting ultrathin-layer polyacrylamide gel isoelectric focusing in the pH range 4-6.5 and neuraminidase pretreated sera. In a Libyan population sample from Tripoli (n = 110) three common phenotypes, alpha 2 HSG 1-1, 2-1, and 2-2, were observed. The allele frequencies were alpha 2 HSG1 = 0.8364 and alpha 2 HSG2 = 0.1636. The theoretical exclusion rate in cases of disputed paternity is 11.8%.     

Further alleles of phosphoglucomutase in human semen detected by isoelectric focusing

A technique for identifying further alleles of PGM1 in human semen detected by isoelectric focusing has been described. A survey of 100 semen samples has shown that there was close agreement between the observed and expected gene frequencies of these new alleles on the basis that there were four common alleles determined by the PGM1 locus and not two as originally proposed [1,2]. The use of these new genetic variatns of PGM will considerably enhance the investigation of semen stains in forensic science.     

A silver staining method for the detection of polymorphic proteins in minute bloodstains after isoelectric focusing

A silver staining method has been developed to study polymorphic proteins in bloodstains after isoelectric focusing. This method is highly sensitive and permits the detection of polymorphic proteins (i.e. alpha 1-antitrypsin, Gc and Tf C subtypes) in bloodstains as small as 0.2 microliter or less. The method is simple and reproducible and can be used after immunofixation. Blood stains can be identified after longer storage periods than is possible by using conventional staining methods.     

Transferrin (Tf) polymorphism: An analysis by isoelectric focusing

The polymorphism of the transferrin (Tf) system was studied in a total of 300 unrelated Japanese individuals from Miyagi prefecture, the northern part of Japan, using isoelectric focusing in thin-layer polyacrylamide gel. In our population samples three common phenotypes and nine variants were observed. The calculated allele frequencies were Tf^C₁ = 0.773, Tf^C₂ = 0.212, Tf^D_chi (Chinese) = 0.008, and the combined frequencies for the Tf^B_var (variants) = 0.007. Family data (n = 44) were in accordance with an autosomal codominant fashion of inheritance. The use of isoelectric focusing procedure among Japanese will raise the probability of excluding a man falsely accused of paternity to 15.8% as compared with 1.5% when the conventional electrophoretic methods are used.     

Orosomucoid 1 phenotyping from human urine by isoelectric focusing

Orosomucoid 1 polymorphism was revealed in human urine by isoelectric focusing and immunoprinting on polyacrylamide gels. The orosomucoid in urine samples was recovered and concentrated by using immobilized allo A lectin. The orosomucoid 1 phenotypes of 60 urine samples correlated with those of the corresponding serum samples.     

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.     

DIA3 phenotyping in human semen and seminal stains by isoelectric focusing

The polymorphism of DIA3 was investigated by isoelectric focusing in semen samples from 235 unrelated Japanese volunteers and patients. Besides the three common phenotypes seven samples of the type 3-1 were observed. However, readable isoenzyme patterns were not demonstrated in semen samples of oligospermia under about 10 X 10(6)/ml sperm cells. The allele frequencies were DIA3*1 = 0.821, DIA3*2 = 0.164, and DIA3*3 = 0.015. The DIA3*1 frequency in oligospermia (0.765) was lower than that in normospermia (0.836). The isoelectric focusing method was successfully applied to phenotyping DIA3 in seminal stains; each phenotype was demonstrated at 37 degrees C for up to 4 weeks, at room temperature for up to 8 weeks, and at 4 degrees C for over 12 weeks after stain formation. In vaginal swabs the isoenzyme bands were very faint and not identifiable.     

A method of human diaphorase locus 3 phenotyping in human semen by isoelectric focusing

A rapid method for the separation of diaphorase (DIA 3) isozymes in the pH range 6-8 is presented. Isoelectric focusing (IEF) of DIA 3 on 0.2 mm thick polyacrylamide gels took 60 min at 5 degrees C. Separated bands are sharp and common phenotypes are readily distinguishable. The method combines high resolution, speed, reproducibility and reagent economy with simplicity.     

The typing of α1-antitrypsin in human bloodstains by isoelectric focusing

A polyacrylamide gel isoelectric focusing (PAGIF) technique is described for the determination of α₁-antitrypsin (Pi) phenotypes in bloodstains. The time limits for Pi type determination of bloodstains kept under different storage conditions are given. The resolution of PAGIF in the typing of Pi phenotypes in human bloodstains in investigated.     

The PGM1 polymorphism as revealed by ultrathin-layer isoelectric focusing in the population of Padua

The occurrence of PGM₁ phenotypes in 589 samples from the population of Padua was investigated by ultrathin-layer isoelectric focusing. All ten phenotypes were observed. Frequencies of the PGM₁ alleles (1+ = 0.6180; 1? = 0.1163; 2+ = 0.2122; 2? = 0.0535) have been compared to those found in other populations.     

Genetic study of red cell esterase D polymorphism by ultrathin layer isoelectric focusing. Distribution in the Veneto population (Italy)

Human red cell Esterase D (EsD) was analyzed by isoelectric focusing (IEF) on ultrathin-layer polyacrylamide gel with a pH range of 5.0-6.0. Hemolysates were treated with Dithiothreitol to avoid loss of activity and change of the isozyme patterns by in vitro storage effects. In our sample of 951 unrelated persons from Veneto, seven different phenotypes were observed. The following allele frequencies were calculated: EsD1 = 0.8476, EsD2 = 0.1336, EsD5 = 0.0178, and EsDV = 0.0010.     

Identification of species specific hemoglobin by isoelectric focusing

A method for the discrimination of human hemoglobin from animal sources is described. Hemoglobins from 34 species with and without treatment by p-chloromercuri-benzoate (PCMB) were examined by thin-layer isoelectric focusing (IEF) in polyacrylamide gel (PAG) plates containing an Ampholine gradient of pH 3.5 – 9.5. PCMB treatment was effective in distinguishing human and animal hemoglobins with their characteristic IEF patterns. Values for the isoelectric points of the major and minor hemoglobins of each species are also described.     

Single-strand conformation polymorphism of hyper-variable regions HV1 and HV2 of human mitochondrial DNA: detection by silver staining

Human mitochondrial DNA from 50 trios consisting of mother (M), child (C) and father (F) was PCR amplified with primers flanking the hyper-variable regions, HVR1 and HVR2. The amplified products were then fractionated under non-denaturing conditions, silver-stained and compared by single-stranded conformational polymorphism (SSCP). In all but one case, mother and child displayed identical patterns, which could be promptly distinguished from that of the father. For the remaining cases, either set of primers was sufficient to resolve the familial ties. In no instance, M displayed alleles different from those of C within each trio, demonstrating that no false exclusions occurred. The SSCP approach proved to be a robust technique suitable as a preliminary screening in cases requiring identification of multiple samples.     

The polymorphism of transferrin by ultrathin-layer isoelectric focusing. Tf phenotypes and TfC subtypes in the population of Padua

The distribution of Tf phenotypes in the population of Padua was investigated by ultrathin-layer isoelectric focusing. In our sample (n = 618) nine phenotypes, Tf C₁, C₂, C₃, C_3?1, C_2?1, C_3?2, C₁B, C₂B and C₁D, were observed and the following frequencies calculated: TfC₁ = 0.77837; TfC₂ = 0.1804; TfC₃ = 0.03641; TfB = 0.0040; TfD = 0.0008. These gene frequencies have been compared to those found in other populations. Analysis of 101 mother-child pairs was in agreement with an autosomal codominant mode of inheritance.     

DIA3 phenotyping in human tissues, dental pulps, and hair roots by isoelectric focusing

The polymorphism of DIA3 was investigated in tissues of various human organs, dental pulps, and hair roots by isoelectric focusing. DIA3 types were demonstrated from tissues of brain, prostate, testis, ovary, and uterus, but not from tissues of spleen, pancreas, heart, liver, muscle, lung, skin, and kidney. Determination was possible from dental pulps stored at room temperature for up to 2 weeks and from fresh hair roots. The results show that the DIA3 typing by isoelectric focusing is useful for medicolegal individualization of brain, reproductive organs, teeth, and hairs.