RESUMO
During an extensive multipopulation study with Y-short tandem repeat (STR) loci, amplified using the AmpFlSTR Yfiler PCR amplification kit, amplification of a 71 bp fragment was observed in 2.32% of the male samples analyzed (N = 3141). By direct sequencing of this fragment, it was determined that the primer binding sequences were identical to those of the DYS456 locus. A T to G single-nucleotide polymorphism (SNP) enabled amplification of the 71 bp fragment. The SNP is located within an X-Y homologous region at Xq21.31 and was observed with the highest frequency within the African American and Sub-Saharan African populations in our study. Presence of SNP on the X chromosome did not interfere with the reliability of typing the DYS456 locus and the other Y-STR loci typeable using the AmpFlSTR Yfiler PCR amplification kit. Full profiles in a mixture of male:female at 1:4000 were obtained using the current configuration of the AmpFlSTR Yfiler kit even in the presence of female DNA containing the G variant.
Assuntos
Cromossomos Humanos X , Impressões Digitais de DNA , Polimorfismo de Nucleotídeo Único , Sequências de Repetição em Tandem , Cromossomos Humanos Y , Primers do DNA , Feminino , Frequência do Gene , Humanos , Masculino , Mutação , Reação em Cadeia da Polimerase , Grupos Raciais/genética , Análise de Sequência de DNARESUMO
In the past 5 years, there has been a substantial increase in the use of Y-short tandem repeat loci (Y-STRs) in forensic laboratories, especially in cases where typing autosomal STRs has met with limited success. The AmpFlSTR Yfiler PCR amplification kit simultaneously amplifies 17 Y-STR loci including the loci in the "European minimal haplotype" (DYS19, DYS385a/b, DYS389I, DYS389II, DYS390, DYS391, DYS392, and DYS393), the Scientific Working Group on DNA Analysis Methods (SWGDAM) recommended Y-STR loci (DYS438 and DYS439), and the highly polymorphic loci DYS437, DYS448, DYS456, DYS458, Y GATA H4, and DYS635 (formerly known as Y GATA C4). The Yfiler kit was validated according to the FBI/National Standards and SWGDAM guidelines. Our results showed that full profiles are attainable with low levels of male DNA (below 125 pg) and that under optimized conditions, no detectable cross-reactive products were obtained on human female DNA, bacteria, and commonly encountered animal species. Additionally, we demonstrated the ability to detect male specific profiles in admixed male and female blood samples at a ratio of 1:1000.
Assuntos
Cromossomos Humanos Y , Impressões Digitais de DNA/métodos , Sequências de Repetição em Tandem , Animais , Gatos/genética , Impressões Digitais de DNA/normas , Primers do DNA , Cães/genética , Genética Populacional , Humanos , Masculino , Pan troglodytes/genética , Reação em Cadeia da Polimerase , Grupos Raciais/genética , Reprodutibilidade dos Testes , Especificidade da EspécieRESUMO
Analysis of length polymorphism at short tandem repeat (STR) loci utilizing multiplex polymerase chain reaction (PCR) remains the primary method for genotyping forensic samples. The AmpFâSTR(®) Identifiler(®) Plus PCR Amplification Kit is an improved version of the AmpFâSTR(®) Identifiler(®) PCR Amplification Kit and amplifies the core CODIS loci: D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, CSF1PO, FGA, TH01, TPOX, and vWA. Additional loci amplified in the multiplex reaction are the sex-determinant, amelogenin, and two internationally accepted loci, D2S1338 and D19S433. While the primer sequences and dye configurations were unchanged, the AmpFâSTR(®) Identifiler(®) Plus PCR Amplification Kit features an enhanced buffer formulation and an optimized PCR cycling protocol that increases sensitivity, provides better tolerance to PCR inhibitors, and improves performance on mixture samples. The AmpFâSTR(®) Identifiler(®) Plus PCR Amplification Kit has been validated according to the FBI/National Standards and Scientific Working Group on DNA Analysis Methods (SWGDAM) guidelines. The validation results support the use of the AmpFâSTR(®) Identifiler(®) Plus PCR Amplification Kit for human identity and parentage testing.
Assuntos
Impressões Digitais de DNA/instrumentação , Reação em Cadeia da Polimerase/instrumentação , Animais , Degradação Necrótica do DNA , Primers do DNA , Eletroforese , Frequência do Gene , Genética Populacional , Genótipo , Humanos , Repetições de Microssatélites , Primatas/genética , Grupos Raciais , Especificidade da EspécieRESUMO
The Quantifiler Duo DNA Quantification kit enables simultaneous quantification of human DNA and human male DNA as well as detection of inhibitors of PCR in a single real-time PCR well. Pooled human male genomic DNA is used to generate standard curves for both human (ribonuclease P RNA component H1) and human male (sex determining region Y) specific targets. A shift in the cycle threshold (C(T)) values for the internal positive control monitors the presence of PCR inhibitors in a sample. The assay is human specific and exhibits a high dynamic range from 0.023 to 50 ng/microL. In addition, the multiplex assay can detect as little as 25 pg/microL of human male DNA in the presence of a 1000-fold excess of human female DNA. The multiplex assay provides assessment of the DNA extract and guidance for the selection of the appropriate AmpFlSTR Amplification Kit to obtain interpretable short tandem repeat profiles.
Assuntos
DNA/análise , Reação em Cadeia da Polimerase/métodos , Animais , População Negra , Cromossomos Humanos Y , Degradação Necrótica do DNA , Impressões Digitais de DNA , Feminino , Genética Populacional , Hemina/administração & dosagem , Hemina/análise , Humanos , Substâncias Húmicas/análise , Masculino , RNA/genética , Reprodutibilidade dos Testes , Ribonuclease P/genética , Sensibilidade e Especificidade , Especificidade da Espécie , Sequências de Repetição em Tandem , População BrancaRESUMO
The PrepFiler Forensic DNA Extraction Kit enables isolation of genomic DNA from a variety of biological samples. The kit facilitates reversible binding of DNA with magnetic particles resulting in high DNA recovery from samples with very low and high quantities of biological materials: 0.1 and 40 microL of human blood (donor 2) provided 14 and 2883 ng of DNA, respectively. Following the revised SWGDAM guidelines, performance of the developed method was investigated using different sample types including saliva on swabs, semen stains on cotton fabric, samples exposed to environment, samples with polymerase chain reaction (PCR) inhibitors, blood stains (on denim, cotton cloth, and FTA paper), and touch evidence-type samples. DNA yields for all samples tested were equal or better than those obtained by both phenol-chloroform extraction and commercial kits tested. DNA obtained from these samples was free of detectable PCR inhibitors. Short tandem repeat profiles were complete, conclusive, and devoid of PCR artifacts.