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1.
Arch Oral Biol ; 110: 104626, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31838295

RESUMO

OBJECTIVE: Dental fluorosis (DF) is a dental development disorder caused by chronic fluoride overconsumption. There are differences in the susceptibility to and severity of DF in studied populations. The objective of the present study was to determine if single-nucleotide variations (SNVs) in the genes Amelogenin (AMELX), Odontogenic Ameloblast Associated (ODAM) and Matrix Metalloproteinase 20 (MMP20) are associated with DF by evaluating the relationship between variations in these genes and the degree of DF severity. SUBJECTS AND METHODS: Schoolchildren from two regions of Durango State and Mexico City, Mexico, were studied. The DF phenotype was determined using the Thylstrup and Fejerskov (TF) index. DNA was obtained from the buccal mucosa of each participant, and the presence of the variations rs946252 in AMELX, rs1514392 in ODAM and rs1784418 in MMP20 was determined by bidirectional DNA sequencing. RESULTS: A total of 180 DNA samples from 30 schoolchildren from 2 areas of Durango State were sequenced and analyzed. Differences in the severity of DF were found between the study areas (p = 0.006). SNVs in theMMP20 gene were present in 76.9 % of the participants in the high fluoride concentration and lower DF severity area. CONCLUSION: AMELX and ODAM variations was not different between the two populations with respect to DF severity; however, the presence of rs1784418 differed between phenotypes with regard to susceptibility to DF. Therefore, MMP20 might be related to the various phenotypes of DF and may serve as a protective marker.


Assuntos
Amelogenina , Fluorose Dentária , Peptídeos e Proteínas de Sinalização Intracelular , Metaloproteinase 20 da Matriz , Amelogenina/genética , Proteínas de Transporte , Criança , Fluoretos , Fluorose Dentária/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Metaloproteinase 20 da Matriz/genética , México , Fenótipo , Análise de Sequência de DNA
2.
Forensic Sci Int Genet ; 43: 102139, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31487605

RESUMO

Correct identification of probative samples is the first crucial step in the analysis of sexual assault kits (SAKs). We report a nucleic acid-based approach, as an alternative to the widely utilized p30 assay, to screening male DNA from SAKs collected from female victims by combining a rapid lysis protocol with an isothermal amplification method. The enzymatic lysis protocol efficiently digests biological material to release nuclear DNA in 10 min in a single closed tube, including resilient cell types such as sperm cells. The amplification and detection of human male specific DNA is achieved through loop-mediated isothermal amplification (LAMP) accompanied with hydroxynaphthol blue, a colorimetric indicator, producing a visually-distinctive color change in the presence of male DNA. The Y-screen approach demonstrated high specificity to human male DNA, can reliably detect target DNA as low as 50 pg, and correctly identified all probative samples from 14 single-blind mock sexual assault samples. In contrast with the widely used p30 assay which requires at least 2 h incubation time and manual application to a lateral flow pad, this Y-screen assay can be completed in half the time, and can be performed in a 96-well format without the need for a fluorescence detector, making facile high-throughput sample screening possible.


Assuntos
Colorimetria , Técnicas de Amplificação de Ácido Nucleico/métodos , Espermatozoides/química , Amelogenina/genética , Cromossomos Humanos Y , DNA/análise , Marcadores Genéticos , Humanos , Indicadores e Reagentes , Masculino , Naftalenossulfonatos , Reação em Cadeia da Polimerase , Delitos Sexuais
3.
J Dairy Sci ; 102(11): 10100-10104, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31447157

RESUMO

Freemartinism is the most common type of disorder of sex development in cattle. It leads to sterility in the female co-twin in heterosexual twin pregnancy, and is thus a serious problem in cattle production. The incidence of freemartin syndrome is directly dependent on the prevalence of twinning, which has increased in dairy cattle populations in recent years. Thus, early and rapid identification of freemartins is needed to reduce economic loss. Of the various methods used to diagnose this condition, identifying the XX and XY cell lines in blood samples using cytogenetic techniques is the gold standard; however, this technique is time consuming. Faster and more reliable techniques are thus being sought. Droplet digital PCR (ddPCR) is a third-generation PCR method and it has not previously been used to detect XX/XY leukocyte chimerism in cattle. The aim of the present study was to verify the usefulness of ddPCR to detect and quantify leukocyte chimerism in this species. The X and Y copy numbers were estimated by identifying the copy numbers of 2 genes located on the sex chromosomes: amelogenin X-linked (AMELX) on the X chromosome and amelogenin Y-linked (AMELY) on the Y chromosome. In the first step, we performed ddPCR on samples prepared from female DNA mixed with male DNA in serially diluted proportions. We determined that the sensitivity of this method was sufficient to detect a low-frequency (<5%) cell line. In the next step, ddPCR was used to analyze 22 Holstein Friesian freemartins. Cytogenetic evaluation of these cases revealed leukocyte chimerism; the proportion of XX and XY metaphase spreads varied over a wide range, from XX (98%)/XY (2%) to XX (4%)/XY (96%). The use of ddPCR facilitated the precise estimation of the ratio of the copy number of X to Y sex chromosomes. In all cases, the XX/XY chimerism detected by cytogenetic analysis was confirmed using ddPCR. The method turned out to be very simple, accurate, and sensitive. In conclusion, we recommend the ddPCR method for fast and reliable detection of XX/XY leukocyte chimerism in cattle.


Assuntos
Amelogenina/genética , Quimerismo/veterinária , Freemartinismo/diagnóstico , Reação em Cadeia da Polimerase/veterinária , Cromossomos Sexuais/genética , Animais , Bovinos , Feminino , Freemartinismo/genética , Leucócitos , Masculino , Reação em Cadeia da Polimerase/métodos , Gravidez , Sensibilidade e Especificidade , Cromossomo X/genética , Cromossomo Y/genética
4.
Forensic Sci Int Genet ; 43: 102147, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31437781

RESUMO

With the continuous development of massively parallel sequencing (MPS), increasing numbers of laboratories have utilized this method for forensic genomic analyses. When sequencing common short tandem repeats (STRs), MPS does have many advantages over the length-based genotyping method that uses traditional capillary electrophoresis (CE) technology. The Precision ID GlobalFiler™ NGS STR Panel v2 was recently released to simultaneously target 31 autosomal STRs (20 expanded Combined DNA Index System (CODIS) core loci and 11 non-CODIS loci) and 4 gender determination loci (Amelogenin, DYS391, SRY and Y-indel (rs2032678)) with the Ion S5™ System. In the current study, we performed a preliminary validation for this novel MPS-STR panel that included the following analyses: repeatability, concordance, stutter and balance, sensitivity, case-type sample testing, stability, mixture and a population investigation. Complete and reliable profiles were obtained using 125 pg of positive control DNA. The commonly encountered types of case samples and artificial mixtures with ratios of 1:1, 1:3 and 3:1 were also fully genotyped. Additional allele sequence variations were detected in samples from 50 unrelated individuals, and subsequently, an increased power of discrimination and power of exclusion were achieved. However, the average depth of coverage (DoC) of the Penta D locus was detected to be dramatically lower than those of other loci, which caused an interlocus imbalance; this could be one of the reasons for the intralocus imbalance of this locus and the 0.18% inconsistent results in the concordance study. Although certain flaws were observed, the informative metrics, including the DoC, sequence coverage ratios (SCRs) and heterozygote balance (Hb), of the novel MPS multiplex in our study were sufficient for reliable sequencing results that were 99.61% in concordance with the capillary electrophoresis (CE) results. In general, the Precision ID GlobalFiler™ NGS STR Panel v2 was demonstrated to be sensitive, reliable and robust and could be a powerful tool for human identification and kinship analyses. Additionally, we look forward to its updated version.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , Repetições de Microssatélites , Análise de Sequência de DNA , Amelogenina/genética , Impressões Digitais de DNA , Grupos Étnicos/genética , Feminino , Genética Forense , Humanos , Masculino , Projetos Piloto , Reação em Cadeia da Polimerase , Reprodutibilidade dos Testes
5.
Sud Med Ekspert ; 62(4): 19-21, 2019.
Artigo em Russo | MEDLINE | ID: mdl-31407701

RESUMO

The amelogenin gene encodes dental enamel protein and is present in humans in two forms - AMELX and AMELY, located on the X- and Y-chromosomes, respectively. This rare case depicts a partial deletion of the AMELY gene. In the Into-Stil LLC laboratory, we performed the genetic testing of the DNA samples extracted from buccal epithelial cells of the alleged father and the disputed child (a boy). Genotyping was carried out using COrDIS Plus ('Gordis', Russian Federation) and AmpFLSTR Identifiler Direct PCR Amplification ('Applied Biosystems', USA) Kits. Our findings have demonstrated that both the alleged father and the disputed child lacked the fragments corresponding with the AMELY gene. Using both STR-systems, we detected, in the disputed child's genome, the allele formally identical to the allele in the genome of the alleged father. Further analysis using the COrDYS ('Gordis', Russian Federation) kit allowed us to detect the amplified fragments corresponding with all the STR loci of Y chromosome, except DYS576 and DYS449, which confirmed that both studied individuals belonged to male biological sex.


Assuntos
Amelogenina/genética , Deleção Cromossômica , Cromossomos Humanos Y/genética , Técnicas de Genotipagem , Paternidade , Criança , Humanos , Masculino , Reação em Cadeia da Polimerase , Federação Russa
6.
Proc Natl Acad Sci U S A ; 116(28): 13867-13872, 2019 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-31239344

RESUMO

Small variations in the primary amino acid sequence of extracellular matrix proteins can have profound effects on the biomineralization of hard tissues. For example, a change in one amino acid within the amelogenin protein can lead to drastic changes in enamel phenotype, resulting in amelogenesis imperfecta, enamel that is defective and easily damaged. Despite the importance of these undesirable phenotypes, there is very little understanding of how single amino acid variation in amelogenins can lead to malformed enamel. Here, we aim to develop a thermodynamic understanding of how protein variants can affect steps of the biomineralization process. High-resolution, in situ atomic force microscopy (AFM) showed that altering one amino acid within the murine amelogenin sequence (natural variants T21 and P41T, and experimental variant P71T) resulted in an increase in the quantity of protein adsorbed onto hydroxyapatite (HAP) and the formation of multiple protein layers. Quantitative analysis of the equilibrium adsorbate amounts revealed that the protein variants had higher oligomer-oligomer binding energies. MMP20 enzyme degradation and HAP mineralization studies showed that the amino acid variants slowed the degradation of amelogenin by MMP20 and inhibited the growth and phase transformation of HAP. We propose that the protein variants cause malformed enamel because they bind excessively to HAP and disrupt the normal HAP growth and enzymatic degradation processes. The in situ methods applied to determine the energetics of molecular level processes are powerful tools toward understanding the mechanisms of biomineralization.


Assuntos
Amelogênese Imperfeita/genética , Amelogenina/genética , Biomineralização/genética , Proteínas da Matriz Extracelular/genética , Adsorção/genética , Amelogênese Imperfeita/metabolismo , Amelogênese Imperfeita/patologia , Amelogenina/química , Sequência de Aminoácidos/genética , Substituição de Aminoácidos/genética , Aminoácidos/química , Aminoácidos/genética , Animais , Durapatita/química , Metabolismo Energético/genética , Proteínas da Matriz Extracelular/química , Humanos , Metaloproteinase 20 da Matriz/química , Metaloproteinase 20 da Matriz/genética , Camundongos , Microscopia de Força Atômica , Conformação Proteica , Termodinâmica
7.
Int. j. morphol ; 37(2): 522-532, June 2019. graf
Artigo em Inglês | LILACS | ID: biblio-1002254

RESUMO

Amelogenin is one of the enamel matrices secreted by ameloblasts. A mutation of the amelogenin gene can cause hereditary dental enamel defects known as amelogenesis imperfecta (AI). Since lysosome-associated membrane protein-1 (LAMP-1), -3 (LAMP-3), and 78kDa glucose-related protein (Grp78) were identified as binding proteins of amelogenin, several studies have suggested the involvement of these binding proteins with the cell kinetics of ameloblasts in normal or abnormal conditions. The purpose of this study is to investigate the distribution of these amelogenin binding proteins in the ameloblast cell differentiation of mice with a point mutation of the amelogenin gene (Amelx*). The incisors of Amelx* mice had a white opaque color and the tooth surface was observed to be rough under a scanning electron microscope. Among the sequential ameloblast cell differentiation in the Amelx* mice, the shape of ameloblasts at the transition stage was irregular in comparison to those in wild-type (WT) mice. Immunostaining of Grp78 revealed that the whole cytoplasm of the transition stage ameloblasts was immunopositive for Grp78 antibody, while only the distal part of cell was positive in the WT mice. Furthermore, in the Amelx* mice, the cytoplasm of the transition stage ameloblasts was immunopositive for LAMP-1 and LAMP-3. These results suggest that Amelx* may cause the abnormal distribution of amelogenin binding proteins in the cytoplasm of ameloblasts.


La amelogenina es una de las matrices de esmalte secretadas por los ameloblastos. Una mutación del gen de amelogenina puede causar defectos hereditarios del esmalte dental conocidos como amelogénesis imperfecta (AI). Dado que la proteína de membrana asociada a lisosoma-1 (LAMP-1), -3 (LAMP-3) y la proteína relacionada con la glucosa de 78 kDa (Grp78) se identificaron como proteína de unión a amelogenina, varios estudios han sugerido la participación de estas proteínas con la cinética celular de los ameloblastos en condiciones normales o anormales. El objetivo del estudio fue investigar la distribución de LAMP-1, LAM-3 y Grp78 durante la diferenciación celular de ameloblastos de ratones con una mutación puntual del gen de amelogenina (Amelx*). Los incisivos de los ratones Amelx* presentaron un color blanco opaco y se observó en microscopio electrónico de barrido que la superficie del diente era áspera. La diferenciación celular secuencial y la forma de los ameloblastos en la etapa de transición en los ratones Amelx* fue irregular en comparación con los ratones silvestres (RS). La inmunotinción de Grp78 reveló que todo el citoplasma de los ameloblastos en etapa de transición fue inmunopositivo para el anticuerpo Grp78, mientras que solo la parte distal de la célula fue positiva en los ratones RS. Además, en ratones Amelx*, el citoplasma de los ameloblastos en etapa de transición fue inmunopositivo para LAMP-1 y LAMP-3. Estos resultados sugieren que Amelx* puede causar distribución anormal de proteínas de unión a amelogenina en el citoplasma de los ameloblastos.


Assuntos
Animais , Camundongos , Glicoproteínas de Membrana Associadas ao Lisossomo/metabolismo , Amelogenina/metabolismo , Amelogênese Imperfeita , Proteínas de Choque Térmico/metabolismo , Microscopia Eletrônica de Varredura , Imunofluorescência , Esmalte Dentário/patologia , Proteína 1 de Membrana Associada ao Lisossomo/metabolismo , Amelogenina/genética , Proteína 3 de Membrana Associada ao Lisossomo/metabolismo , Incisivo/patologia
8.
J Genet ; 982019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30945688

RESUMO

Yak, an economically important bovine species considered as lifeline of the Himalaya. Indeed, this gigantic bovine is neglected because of the scientific intervention for its conservation as well as research documentation for a long time. Amelogenin is an essential protein for tooth enamel which eutherian mammals contain two copies in both X and Y chromosome each. In bovine, the deletion of a fragment of the nucleotide sequence in Y chromosome copy of exon 6 made Amelogenin an excellent sex-specific marker. Thus, an attempt was made to use the gene as an advanced molecular marker of sexing of the yak to improve breeding strategies and reproduction. The present study confirmed that the polymerase chain reaction amplification of the Amelogenin gene with a unique primer is useful in sex identification of the yak. The test is further refined with qPCR validation by quantifying the DNA copy number of the Amelogenin gene in male and female. We observed a high level of sequence polymorphisms of AMELX and AMELY in yak considered as novel identification. These tests can be further extended into several other specialized fields including forensics, meat production and processing, and quality control.


Assuntos
Amelogenina/genética , Genoma , Genômica/métodos , Polimorfismo Genético , Análise para Determinação do Sexo/métodos , Animais , Sequência de Bases , Bovinos , Feminino , Masculino , Reação em Cadeia da Polimerase
9.
Genet Test Mol Biomarkers ; 23(5): 359-362, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30994363

RESUMO

Aim: The amelogenin gene is a widely used gender marker for forensic DNA profiling. Males who have the amelogenin Y (AMELY) allele deletion can be mistakenly identified as females if genotyping is performed only on the amelogenin gene. The aim of this study was to investigate the frequency of the AMELY allele deletion in the Chinese Han population and analyze the possible genetic variation on the Y chromosome. Materials and Methods: The amelogenin gene of 12,735 unrelated males from the Chinese Han population were genotyped using common forensic short tandem repeat (STR) kits. The AMELY allele deletion was verified by redesigned primers and sequencing. Eighteen Y-specific sequence tagged sites (STSs) on the Yp11.2 region were selected to delineate the deletion breakpoints on the Y chromosome. Results: Three males were confirmed to have no AMELY allele. The frequency rate of the AMELY-null allele was 0.236% (3/12,735) in the Chinese Han population of Central China; 2.73 Mb of sequence on the Y chromosome were absent in all the AMELY-negative samples. The deleted region was mapped using SRY, AMELY, 5 Y-STRs, and 18 STSs, which belong to the class I deleted pattern. The three unrelated males shared the same Y-STR haplotype with four males from other Chinese populations, all of whom have the AMELY-null allele. The haplogroup of these males was identified as the O3 haplogroup. Conclusion: The AMELY allele deletion in the Chinese population was accompanied by the deletion of the Y-STR loci on the Yp11.2 region. Therefore, another Y-specific marker should be tested simultaneously when unknown samples are examined as part of a criminal investigation.


Assuntos
Amelogenina/genética , Adulto , Alelos , Amelogenina/metabolismo , Amelogenina/fisiologia , Grupo com Ancestrais do Continente Asiático/genética , China , Cromossomos Humanos Y/genética , Impressões Digitais de DNA/métodos , Análise Mutacional de DNA/métodos , Grupos Étnicos/genética , Frequência do Gene/genética , Genótipo , Haplótipos , Humanos , Masculino , Repetições de Microssatélites , Fenótipo , Deleção de Sequência/genética , Análise para Determinação do Sexo/métodos
10.
Electrophoresis ; 40(12-13): 1662-1676, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31012482

RESUMO

We have developed a novel STR 25-plex florescence multiplex-STR kit (DNATyper25) to genotype 23 autosomal and two sex-linked loci for forensic applications and paternity analysis. Of the 23 autosomal loci, 20 are non-CODIS. The sex-linked markers include a Y-STR locus (DYS391) and the Amelogenin gene. We present developmental validation studies to show that the DNATyper25 kit is reproducible, accurate, sensitive, and robust. Sensitivity testing showed that full profiles were achieved with as low as 125 pg of human DNA. Specificity testing demonstrated a lack of cross reactivity with a variety of commonly encountered non-human DNA contaminants. Stability testing showed that full profiles were obtained with humic acid concentration ≤60 ng/µL and hematin concentration <400 µM. For forensic evaluation, the 23 autosomal STRs followed the Hardy-Weinberg equilibrium. In an analysis of 509 Chinese (CN) Hans, we detected a combined total of 181 alleles at the 23 autosomal STR loci. Since these autosomal STRs are independent from one another, PM was 8.4528 × 10-22 , TDP was 0.999 999 999 999 999 999 999, CEP was 0.999 999 8395. The forensic efficiency parameters demonstrated that these autosomal STRs are highly polymorphic and informative in the Han population of China. We performed population comparisons and showed that the Northern CN Han has a close genetic relationship with the Luzhou Han, Tujia, and Bai populations. We propose that the DNATyper25 kit will be useful for cases where paternity analysis is difficult and for situations where DNA samples are limited in quantity and low in quality.


Assuntos
Genética Forense/métodos , Repetições de Microssatélites/genética , Reação em Cadeia da Polimerase/métodos , Amelogenina/genética , Animais , China , Cromossomos Humanos Y/genética , DNA/análise , DNA/classificação , DNA/genética , Técnicas de Genotipagem/métodos , Humanos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
12.
Gac Med Mex ; 155(1): 101-107, 2019.
Artigo em Espanhol | MEDLINE | ID: mdl-30799455

RESUMO

Amelogenesis imperfecta is a group of developmental disorders of the dental enamel that is mainly associated with mutations in the AMELX gene. Clinically, it presents different phenotypes that affect the structure and function of dental enamel both in primary and secondary dentition. The purpose of this study was to conduct a literature review on the AMELX functions and mutations that are related to amelogenesis imperfecta. A literature search was carried out in two databases: PubMed and Web of Science, using the keywords "AMELX", "amelogenin", "amelogenesis imperfecta" and "AMELX mutation". Forty articles were reviewed, with AMELX being found to be the predominant gene in the development of dental enamel and amelogenesis imperfecta by altering the structure of amelogenin. In the past few years, the characteristics of the amelogenesis imperfecta process have been described with different phenotypes of hypoplastic or hypo-mineralized enamel, and different mutations have been reported, by means of which the gene sequencing and the position of mutations have been determined.


Assuntos
Amelogênese Imperfeita/genética , Amelogenina/genética , Esmalte Dentário/patologia , Amelogênese Imperfeita/patologia , Humanos , Mutação , Fenótipo
13.
Forensic Sci Int Genet ; 40: 64-73, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30776773

RESUMO

In this study, a multiplex amplification system including 47 autosomal InDels, 2 Y-chromosome InDels, and the sex-determining marker (Amelogenin) was developed with six fluorescent dyes labeling. These InDels were selected from the previous study based on a series of criteria (0.3 < MAF < 0.5, HET > 0.4, etc). The system was designated the AGCU InDel 50 kit and was validated in a series of studies, including a degradation study; tests for sensitivity, species specificity, reproducibility, stability, applicability to case samples, balance of peak height, and PCR conditions; and a population study. The results showed that AGCU InDel 50 kit was quite sensitive, specific, stable in several PCR conditions or exposure to PCR inhibitors, especially against degradation. 74 case samples and 50 paternity cases with STR mutation events were tested using PowerPlex® 21 System, AGCU InDel 50 kit, and Investigator DIPplex kit, and the results showed that the ratio of loci detected with the developed kit were close to Investigator@ DIPplex kit, but considerably higher than PowerPlex® 21 System for case samples containing low amounts of degraded DNA. As for 50 paternity cases, no mutation was observed in any InDels locus, and the CPIs based on 47 autosomal InDels contained in the AGCU InDel 50 kit were all higher than those based on 30 InDels contained in Investigator® DIPplex kit, except 3 cases. In the population study, 203 unrelated individuals from the Guangdong Han population were detected using the AGCU InDel 50 kit, and the values of combined power of discrimination and combined power of exclusion were 0.999 999 999 999 999 and 0.9997, respectively. Thus, AGCU InDel 50 kit is suitable for individual identification and as a supplemental tool for paternity testing. It is reproducible, accurate and robust for forensic applications and human genetic studies.


Assuntos
Impressões Digitais de DNA , Mutação INDEL , Reação em Cadeia da Polimerase Multiplex/instrumentação , Alelos , Amelogenina/genética , Animais , Cromossomos Humanos Y , Degradação Necrótica do DNA , Corantes Fluorescentes , Genética Populacional , Humanos , Repetições de Microssatélites , Reprodutibilidade dos Testes , Especificidade da Espécie
15.
J Forensic Leg Med ; 61: 108-114, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30553228

RESUMO

INTRODUCTION: Short Tandem Repeats (STRs) are defined as short lengths of 2-7 base pairs spreading through human genome which due to their highly diverse individually distribution are widely applied for identity detection and other forensic medicine purposes. Burdening considerable costs by the conventional methods such as capillary electrophoresis, we aimed to compare concomitant usage of multiplex PCR and denaturing high-performance liquid chromatography (DHPLC) as cheap, fast, highly accurate, and more accessible methods, with capillary electrophoresis (CE) to evaluate their potential for early screening of STRs. MATERIALS AND METHODS: The present study randomly included 20 blood samples from the subjects referred to forensic medicine of Semnan, Iran. According to the size and allele frequency, we selected 8 major STR loci including CSF1PO, VWA, D18S51, TPOX, Amelogenin, FGA, SE33, and Penta D. A quad-STR multiplex PCR was performed for each locus and the PCR products were then analyzed using DHPLC machine and compared with the basic genetic properties obtained by capillary electrophoresis. RESULTS: By optimizing the PCR and DHPLC conditions, our findings suggest this strategy as an effective method for STR detection. The genotypes were determined using size of loci which led to comparable results with capillary electrophoresis confirming an insignificant variation in the detection of TOPX, Amelogenin, CSF1PO, and D18S5 (p = 0.331), but discrepant results for FGA and VWA loci (p = 0.002). CONCLUSION: Our study proposed DHPLC method as an effective screening method to characterize TOPX, Amelogenin, CSF1PO, and D18S51 as frequently used STR loci during identity detection in forensic medicine.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Impressões Digitais de DNA , Repetições de Microssatélites , Amelogenina/genética , Eletroforese Capilar , Humanos , Reação em Cadeia da Polimerase Multiplex
16.
Fa Yi Xue Za Zhi ; 34(4): 396-400, 2018 Aug.
Artigo em Inglês, Chinês | MEDLINE | ID: mdl-30465406

RESUMO

OBJECTIVES: To observe and analyse the Amelogenin allelic loss in parent-child identification cases, and to explore the type and mechanism of Amelogenin allelic loss as well as its influence on gender identification and solutions. METHODS: After the detection by SiFaSTR™ 23plex DNA identification system, samples had the characteristics of the peak area of Amelogenin X was the same as the one of adjacent heterozygote or lower than one half of adjacent homozygote in females while Amelogenin X loss was observed in males were selected. X chromosome STR (X-STR) typing and Amelogenin X sequencing were performed. The samples with Amelogenin Y loss in males were confirmed by the detection of Y chromosome STR typing and sex-determining region of Y (SRY). The type and rate of Amelogenin allelic loss were confirmed and calculated, and the mechanism and influence of this variation were also analysed. RESULTS: Amelogenin X allelic loss was observed in one male sample, the mutation in primer-binding region was confirmed by sequencing. The suspected Amelogenin X allelic loss was observed in four female samples, but the mutation in primer-binding region was confirmed by sequencing in only one sample. Amelogenin Y allelic loss was observed in seven male samples, SRY positive cases was detected in five of them, and two were SRY negative. Y-STR type was detected in four cases of the five SRY positive cases, which was not detected in the two SRY negative cases. The rate of Amelogenin allelic loss was about 0.029%. CONCLUSIONS: Amelogenin X allelic loss does not affect the gender identification, but Amelogenin Y allelic loss may cause wrong gender identification. Thus, Y-STR or SRY should be detected for gender confirmation. When Y-STR genotypes are not detected in a "male" whose SRY detection is also negative, then the chromosome karyotype analysis and sex differentiation related genes test should be taken to further confirm the gender.


Assuntos
Amelogenina/genética , DNA/genética , Perda de Heterozigosidade/genética , Feminino , Humanos , Masculino , Análise para Determinação do Sexo
17.
Leg Med (Tokyo) ; 35: 77-79, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30286361

RESUMO

Hairs are often used for DNA analysis in criminal investigations. DNA analysis of hairs with root sheaths is easy in many cases, but analyses using only the shaft or tip of the hair are often difficult. Here we describe a suspected case of child abuse in which we were commissioned to perform DNA analysis. Among 100 hairs, PCR amplification was succeeded in 99 samples, and as a result of direct sequencing, mitochondrial DNA (mtDNA) of the 99 hairs were classified into 6 types. The most common type was the 8-base substitution type of 16,168T-16,172C-16,183C-16,189C-16,217C-16,249C-16,325C-16,390A, which was observed in 86 hairs. This corresponded to the type of the victim. Total 736 STRs (75.5%) in 975 loci of 65 hairs could be typed, and only an amelogenin locus was typed in another hair. All 15 loci were typed in 10 hairs. STR types of 65 (98.5%) in 66 hairs were consistent with that of the victim. From 10 naturally-shed hair of a person, only 37 STRs (30.8%) in 120 loci of 8 hairs were typed, and all 15 loci could not be typed in these hairs. This difference in success rates of STR analysis suggested strongly that the hairs in this case were not shed naturally but forcibly, and the relevance to child abuse was suspected.


Assuntos
Maus-Tratos Infantis/diagnóstico , DNA Mitocondrial/classificação , DNA/análise , Medicina Legal/métodos , Cabelo/química , Repetições de Microssatélites/genética , Abuso Físico , Acidentes por Quedas , Amelogenina/genética , Pré-Escolar , Evolução Fatal , Feminino , Loci Gênicos/genética , Cabelo/fisiologia , Humanos , Reação em Cadeia da Polimerase/métodos
18.
Indian J Dent Res ; 29(4): 470-476, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30127199

RESUMO

Introduction: Forensic odontology necessarily involves the application of dentistry along with various other branches of sciences which deals with proper handling, examination, evaluation, and presentation of dental evidences, that aids to investigate a crime and deliver justice. Sex determination is a part of forensic odontology and an essential priority when traditional identification of the deceased becomes impossible. Aim: To determine Sex by analysis of the Amelogenin gene using Polymerase Chain Reaction (PCR) method on Deoxyribose nucleic acid (DNA) isolated from dental pulp, which was exposed to various environmental conditions created artificially to mimic a forensic scenario. Materials and Method: This in-vitro study was conducted by subjecting extracted teeth to various conditions imitating a forensic scene, viz. desiccation at room temperatures, immersion in salt water, burial in soil and even exposing to extremes of temperatures. DNA was extracted from dental pulp tissue and sex determination was achieved by amplification of the amelogenin gene through AMEL gene based primers in PCR. Result: Among all the samples used in this study, DNA could be extracted from all, except from those that were subjected to a temperature of 350 °C. DNA amplification and sex determination of the samples were found to be accurate when compared to sex of the individual which was recorded initially, during collection of teeth samples. Conclusion: This study shows teeth to be a potent source of DNA even in extreme environmental conditions, barring high temperatures and determination of sex by PCR amplification of AMEL markers to be quite reliable.


Assuntos
Amelogenina/genética , DNA/genética , DNA/isolamento & purificação , Polpa Dentária , Amplificação de Genes , Reação em Cadeia da Polimerase/métodos , Análise para Determinação do Sexo/métodos , Meio Ambiente , Feminino , Humanos , Masculino
19.
Forensic Sci Int Genet ; 37: 73-80, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30103145

RESUMO

Forensic human identification (HID) laboratories occasionally encounter non-specific peaks generated by non-human DNA. Casework samples for human short tandem repeat (STR) profiling may be contaminated by animal DNA because of the specific environment or situation from which they were obtained. Validation studies for HID kits have reported that non-specific peaks generated from some animals are observed near the human amelogenin peak. In this study, we first revealed that DNA sequences associated with the non-specific peaks generated from animal DNA differ from one animal family to the other. However, non-specific peaks cannot be analyzed using the remainder of polymerase chain reaction (PCR) products left over from conventional HID kits when human and animal DNA are mixed. To overcome this issue, we have developed a novel analysis method of using non-specific peaks generated from animal DNA in human STR profiling to identify the source of contaminating animal DNA at the family level. The method applied here is termed as blocking PCR, which involves selective animal DNA re-amplification by blocking nontarget human amelogenin DNA amplification using an oligonucleotide probe that specifically binds to human amelogenin using the remaining PCR product from the HID kit. Our data demonstrated that HID and family discrimination among animals that are often encountered in forensic contexts could be performed simultaneously. This study enabled recovery of more information from limited quantities of casework samples contaminated with animal DNA, which would be useful for forensic HID scientists.


Assuntos
Amelogenina/genética , Impressões Digitais de DNA/métodos , Repetições de Microssatélites , Reação em Cadeia da Polimerase/métodos , Análise de Sequência de DNA , Animais , Cromossomos Humanos X , Cromossomos Humanos Y , Contaminação por DNA , Humanos , Sondas de Oligonucleotídeos , Especificidade da Espécie , Espectrofotometria
20.
PLoS One ; 13(7): e0200700, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30020969

RESUMO

Like DIP-STR markers (deletion/insertion polymorphism-short tandem repeat combinations), SNP-STR markers (single nucleotide polymorphism-STR combinations) are also valuable in forensic DNA mixture analysis. In this study, eight SNP-STRs were selected, and a stable and sensitive multiplex polymerase chain reaction (PCR) assay was developed for amplifying these SNP-STRs and the Amelogenin gender marker according to the principle of amplification refractory mutation system (ARMS). This novel multiplex set allows detection of the minor DNA contributor in a DNA mixture of any gender and cellular origin with high resolution (beyond a DNA ratio of 1:20). In addition, SNP-STR haplotype frequencies were estimated based on a survey of 350 unrelated individuals from Chinese Han population, and the combined power of discrimination (PD) and power of exclusion (PE) of the eight SNP-STRs were calculated as 0.99999999965 and 0.9996, which were obviously higher than that of the eight STR loci: 0.9999999954 and 0.9989 respectively. The results indicated that the SNP-STR compound markers have higher application value in forensic identification compared to standard autosomal STRs, especially in the analysis of imbalanced DNA mixtures.


Assuntos
Amelogenina/genética , Genética Forense/métodos , Reação em Cadeia da Polimerase Multiplex/métodos , Polimorfismo de Nucleotídeo Único , Adulto , Grupo com Ancestrais do Continente Asiático , China , Feminino , Marcadores Genéticos , Humanos , Masculino
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