Measuring human DNA degradation and gender detection in forensic DNA samples by q-PCR/HRM analysis
Human DNA quantification, DNA degradation assessment and gender determination are key aspects in most field of human DNA analysis. The assay reported here is a tri-plex Real Time quantitative PCR reaction followed by high resolution melting (HRM) using Syto9 as intercalating dye. The system produces...
| Autores: | , , , |
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| Tipo de recurso: | artículo |
| Estado: | Versión publicada |
| Fecha de publicación: | 2017 |
| País: | Argentina |
| Institución: | Consejo Nacional de Investigaciones Científicas y Técnicas |
| Repositorio: | CONICET Digital (CONICET) |
| Idioma: | inglés |
| OAI Identifier: | oai:ri.conicet.gov.ar:11336/48625 |
| Acceso en línea: | http://hdl.handle.net/11336/48625 |
| Access Level: | acceso abierto |
| Palabra clave: | Degraded Dna Assessment Dna Quantitation High Resolution Melting Analysis Male Dna Detection https://purl.org/becyt/ford/1.6 https://purl.org/becyt/ford/1 |
| Sumario: | Human DNA quantification, DNA degradation assessment and gender determination are key aspects in most field of human DNA analysis. The assay reported here is a tri-plex Real Time quantitative PCR reaction followed by high resolution melting (HRM) using Syto9 as intercalating dye. The system produces three amplicons: 1- transducin (beta)-like 1, Y-linked −TBL1Y (84 bp), 2- DeGraded small target DNA–DGst- (152 bp) and 3-DeGraded large target DNA-DGlt- (244 bp). DNA quantitation is based on total fluorescence; TBL1Y amplicon allows detecting male DNA and the ratio DGst/DGlt to assess DNA degradation level. q-PCR quantitation proved good linearity in triplicates among 3.2 pg/ul–50 ng/ul DNA concentration range. Amplification efficiency (E) and reaction slope (m) mean values were 1.04 and 3.23 respectively. Upon HRM analysis, three melting peaks are detected in a male DNA sample and two if only female DNA is present. We define the parameter D as the ratio DGst/DGlt that reflects the extent of DNA degradation in a given sample. A direct correlation has been demonstrated between DNA damage and increased value of parameter D. This q-PCR approach is rapid, sensitive, and a cost-effective method suitable for detecting degraded DNA samples and applicable to any field where human DNA quantitation-qualification is required. |
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