The aim of this study was to validate the sex typing based on amplification of X-Y homologous Amelogenin locus in Korean including mutation rate in this locus. It was found that there was no case with reported mutation that may hinder the exact sex typing among 240 Koreans, and the sex typing was successful even with subnanogram quantities of male and female DNA. There was no difference in the sensitivity of reaction among male and female. Differential amplification between X and Y amelogenin bands in some samples was noted, and dilution study revealed that this phenomenon was more frequent when the quantity of sample was low, usually less than 10 ng. That phenomenon was variable between amplification reactions, and was also dependent on different Taq enzyme used for the amplification. When there was differential amplification, the intensity ratio (Y band/X band) ranged about 0.68 - 0.87.
Amelogenin* ; DNA ; Female ; Humans ; Male ; Mutation Rate

OBJECTIVETo establish the expression and purification route for the gene encoding human amelongenin (AMG) mature peptide in Escherichia coli (E. coli).

METHODSRecombined plasmid pGEX-4T-1/AMG was identified by double endonuclease digestion electrophoretogram and DNA sequence analysis. The recombined plasmid was transformed to E. coli BL21. The inducing time, isopropyl-beta-D-thiogalactopyranoside (IPTG) concentration and inducing temperature were optimized for the express system. Under the optimized condition, the target fusing protein in superatant, periplasm, plasm and inclusion body was analyzed separately. A great amount of target fusing protein was found in the dissoluble protein. AMG fusing protein was purified by the GSTrapFF affinity column.

RESULTSDouble endonuclease digestion electrophoretogram and DNA sequence analysis were done to identify the recombined vector pGEX-4T-1/AMG. The results were consistent with the anticipation. The optimum inducing time was 14.5 hours. The optimum IPTG concentration was 1.0 mmol/L. The optimum inducing temperature was 20 degrees C. Under this condition, the target protein was expressed to a maximum. Plentiful target protein was expressed in plasm and inclusion body under the optimized condition. A mount of plasm protein was obtained and purified by the GSTrapFF affinity column. The purified liquid was collected and analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PACE). The protein electrophoresis map showed that AMG fusing protein was purified successfully. After twice elution, high pure fusing protein was obtained.

CONCLUSIONpGEX-4T-1/AMG system is used successfully to express human AMG fusing protein.

OBJECTIVES: To investigate the dynamic process of the self-assembly behaviors of a full-length human amelogenin (AM) and its functional fragments tyrosine-rich amelogenin peptide (TRAP) and leucine-rich amelogenin peptide(LRAP) METHODS: The full-length human AM and its functional fragments, TRAP and LRAP, were reassembled and purified RESULTS: When pH=8, the full-length human AM and TRAP assembly started spontaneously and formed "nanospheres" after 15 min.The nanospheres formed by TRAP existed independently, with a uniform size but without obvious internal structures. The full-length AM was assembled hierarchically, which formed "nanospheres" and further extended in all directions, formed a chain structure, and then aggregated into a net. The self-assembly behavior of LRAP was not obvious. Proteins mostly existed in the form of monomers without "nanosphere" formation. Only few oligomers were observed. The full-length AM was induced independently for 3 days to form rod-shaped HA crystals. TRAP and LRAP proteins were added, after 3 days the crystal elongation was obvious in the c axis, but the growth in plane A and plane B was poor. CONCLUSIONS The self-assembly and mineralization behaviors of full-length human AM, TRAP, and LRAP were consistent with the directional growth mechanism of HA crystals
Amelogenin ; Dental Enamel Proteins ; Durapatite ; Humans

The Y-chromosome, as with other chromosomes in the cell, is subject to mutations. However, unlike autosomal genes, the Y chromosome does not undergo recombination, and therefore individuals from different geographical regions may have differing distribution patterns with respect to Y-chromosome mutations. More detailed knowledge and information regarding Y-chromosome mutations might therefore provide insights into phylogenetic history and personal identification. Here, we describe a case study involving genotype-phenotype discrepancy in an Indian male individual. We found that the mistyping in sex determination was caused by a deletion in the amelogenin Y (AMEL Y) gene. Furthermore, on examining the short tandem repeat (Y-STR) loci using the PowerPlex(R) Y23 System, we found four more deleted loci on Yp11.2 (DYS576, DYS481, DYS570, and DYS458) in this sample. We performed deletion mapping for this sample, and we propose that the microdeletion on the Yp11.2 locus occurred approximately in the 6.44 Mb to 9.75 Mb region. Previous studies have reported that the AMEL Y deletion is a common mutation in the Indian population. Taking into account regional differences, we also analyzed several area-specific Y-chromosome mutations.
Amelogenin ; DNA* ; Humans ; Male ; Microsatellite Repeats ; Recombination, Genetic ; Y Chromosome*

This paper described a collaborative exercise intended to see what kinds of short tandem repeat (STR) loci are used in different DNA typing laboratories in Korea and to compare their results for the demonstration whether uniformity of DNA profiling results from different laboratory could be achieved in Korea. Laboratories were asked to test five tissue DNAs using methods routinely used in each laboratory and to report the results to the coordinating laboratory. The exercise demonstrated that each laboratory was using different STR loci for the typing with different STR numbers, 2 VNTRs, 36 STRs and amelogenin in total, and the direct comparison of the results from all the laboratory for the 18 loci could not be done as only one laboratory submitted typing results. Among 21 loci for which several laboratories submitted typing results, results for 14 loci were the same and results for the other 7 loci were different depending on the participating laboratory. D1S80, F13A01, D16S539, D21S11, D18S51, D3S1744 were the loci with different typing results. Even in the cases where commercial kits were used, the results were not the same depending on the machines used, that is the capillary electrophoresis or the gel based electrophoresis. The reason for the different results, points about the standardization of the methods and the profiling data were described.
Amelogenin ; DNA ; DNA Fingerprinting ; Electrophoresis ; Electrophoresis, Capillary ; Korea ; Microsatellite Repeats

This paper described a collaborative exercise intended to see what kinds of short tandem repeat (STR) loci are used in different DNA typing laboratories in Korea and to compare their results for the demonstration whether uniformity of DNA profiling results from different laboratory could be achieved in Korea. Laboratories were asked to test five tissue DNAs using methods routinely used in each laboratory and to report the results to the coordinating laboratory. The exercise demonstrated that each laboratory was using different STR loci for the typing with different STR numbers, 2 VNTRs, 36 STRs and amelogenin in total, and the direct comparison of the results from all the laboratory for the 18 loci could not be done as only one laboratory submitted typing results. Among 21 loci for which several laboratories submitted typing results, results for 14 loci were the same and results for the other 7 loci were different depending on the participating laboratory. D1S80, F13A01, D16S539, D21S11, D18S51, D3S1744 were the loci with different typing results. Even in the cases where commercial kits were used, the results were not the same depending on the machines used, that is the capillary electrophoresis or the gel based electrophoresis. The reason for the different results, points about the standardization of the methods and the profiling data were described.
Amelogenin ; DNA ; DNA Fingerprinting ; Electrophoresis ; Electrophoresis, Capillary ; Korea ; Microsatellite Repeats

Determination of male and female is important in anthropology, archeology and forensic science. This study was designed to compare genotype sex of improved amelogenin PCR amplication method with morphological sex of ancient human bones. Sixty human skulls which lived from the Bronze Age to twenties centuries and excavated in Uzbekistan were used in this study. Morphological sex was determined by Uzbekistan scientist, and genotype sex was determined by improved amelogenin PCR amplication developed in this study. Among 20 morphological males, 13 samples (65%) were genotypical male. Among 40 morphological females, 20 samples (50%) were genotypical male. In conclusion, morphological method might be inadequate for sex determination of ancient bones. The improved amelogenin PCR method will be useful in sex determination of ancient bones.
Amelogenin* ; Anthropology ; Archaeology ; Female ; Forensic Sciences ; Genotype* ; Humans ; Male ; Polymerase Chain Reaction* ; Skull ; Uzbekistan*

Theoretically, if the odontoblastic layer on the pulpal surface of the dentin is removed, it is impossible to find nucleus after the teeth has undergone endodontic therapy. So dentin does not contain genomic DNA. But it is in existence the possibility that blood permeates into the dentin by endodontic therapy and trauma. Therefore DNA in blood can be detected from the dentin layer. The purpose of this investigation is to evaluate the possibility of individual identification after detection of DNA in the dentin of endodontic created teeth. The authors isolated the human DNA from 40 endodontic treated teeth, performed AmpFLPs by PCR and electrophoresed for detection of STR loci F13A01, LPL gene and X-Y homologous amelogenin gene. The following results were obtained: 1) DNA extraction was possible in 19 endodontic treated teeth out of 40. 2) Sex determination was possible in 12 endodontic treated teeth out of 40 by detection of X-Y homologous amelogenin gene 3) F13A01 locus was detected in 6 endodontic treated teeth out of 40, observed 4 alleles(3.2, 4, 5, 6) and 5 genotypes(3.2-3.2, 3.2-4, 3.2-5, 4-5, 6-6). 4) LPL locus was detected in 7 endodontic treated teeth out of 40, observed 3 alleles(10, 11, 12) and 3 genotypes(10-10, 10-12, 11-12). From the above results DNA extraction, sex determination, amplification of STR locus F13A01, and LPL gene were possible in the endodontic treated teeth and it was suggested that endodontic treated teeth were useful and applicatable as molecular biological samples for individual identification.
Amelogenin* ; Dentin* ; DNA ; Humans ; Odontoblasts ; Polymerase Chain Reaction ; Tooth ; Tooth, Nonvital*

DNA quantification is important to ensure the consistency and the reliability in the interpretation of degraded low copy number DNA typing. We applied the simple PCR quantification method using fluo-rescently labeled primers for the amplification of mtDNA and amelogenin gene in 50 year old skeletal remains (e.g. bone and tooth). K562 DNA was serially diluted and used as a standard for concentration marker to gauge the amount of DNA from PCR versus the peak area. The quantities of DNA extracted from bones and teeth did not show significant difference in the analyses both using mtDNA and amelo-genin gene as an amplification target. To test the efficiency of DNA profiling of degraded low copy number DNA samples, mtDNA PCR quality evaluation and DNA typing for 16 autosomal STR and 9 Y chromosomal STR loci were per-formed and the correlation between DNA quantities and PCR amplification efficiencies of the samples was analyzed. The DNA quantities assayed by the simple method suggested in the present study could be good indicator for mtDNA and STR analysis. As the allele drop-out was observed in less than 0.050ng DNA samples, at least 0.100ng of DNA is required to produce informative STR profiles. Also, STRs with less than 200bp amplification sizes produce efficient DNA profiles in most cases. Therefore, the develop-ment of mini-STRs with less than 200bp amplification sizes is expected to improve DNA typing in degraded low copy number DNA. Y-STRs are easy to detect allele drop-out or drop-in, and accordingly the efficiency test of Y-STRs as well as autosomal STRs for profiling of degraded low copy number DNA samples is thought to be important.
Alleles ; Amelogenin ; DNA Fingerprinting* ; DNA* ; DNA, Mitochondrial ; Humans ; Middle Aged ; Polymerase Chain Reaction ; Tooth

Sex typing may become the start point in investigations that are usually performed through amelogenin typing. In cases involving genotype-phenotype discrepancy, amelogenin typing could yield misleading results. The rare XX male syndrome is characterized by a phenotypic male with a 46, XX female karyotype. In this point, this case report would help understand the importance of genotype-phenotype discrepancy.
Amelogenin ; Female ; Genes, sry ; Humans ; Karyotype ; Klinefelter Syndrome ; Male ; Y Chromosome