Improving the efficiency of Y-chromosome detection and the quality of STR typing in forensic casework with an in-house made qPCR and HRM system based on SYTO™ 9 chemistry.

DNA quantification prior to STR amplification is a crucial step in forensic casework. Obtaining good-quality genetic STR profiles depends mainly on the amount and integrity of the DNA input in the PCR. In addition, the detection of male trace DNA provides key information for forensic investigation. AIM: To evaluate the correlation between the quantification results obtained with the previously developed Amel-Y system, and its ability to detect Y-chromosome DNA by HRM, with the resulting STR profiles, and to ultimately show that Amel-Y can be routinely used in forensic casework to improve STR and Y-STR results. MATERIAL & METHODS: Biological samples derived from forensic casework (85 reference and 391 evidence samples) were quantified by the Amel-Y system (a duplex qPCR/HRM based on SYTO™ 9 chemistry) using Rotor-Gene 6000. STRs were amplified and analyzed with GeneAmp™ PCR System 9700 or Veriti™ Thermal Cyclers and ABI 3500 Genetic Analyzer, respectively. RESULTS: After DNA normalization, a total of 386 STR profiles were obtained (305 full and 81 partial). Sex typing by HRM was 100% successful in reference samples. Male DNA was detected by HRM in 210 evidence samples. 80/201 were mixed with an excess of female DNA. In addition, Amel-Y was able to detect Y-chromosome DNA in mixed samples that did not amplify the Y-variant of Amelogenin marker with commercial STR kits. The reproducibility and precision of the Amel-Y system were demonstrated (CVCt% ≤ 9.55) within the dynamic range analyzed (0.016-50 ng/µL; 41 independent runs). Amel-Y also proved to be compatible with other real-time PCR platforms. CONCLUSION: We demonstrated that Amel-Y is a robust quantification system that can be routinely used in forensic casework to obtain reliable autosomal STR profiles and can be suitable as a predictor for Y-STR typing success when male DNA is detected. HRM can be used as a rapid screening tool for male DNA detection in mixed samples. Alternative designs like Amel-Y offer independence from commercial quantification kits in forensic labs.

Human DNA degradation assessment and male DNA detection by quantitative-PCR followed by high-resolution melting analysis.

We developed a q-PCR technique that simultaneously evaluates the extent of degradation and determines the gender of a human DNA donor. QYDEG HRM is a triplex real-time PCR whose products are analysed by high-resolution melting (HRM). The system produces three amplicons: (1) transducin (beta)-like 1, Y-linked (TBL1Y) (84bp); (2) large-target sequence (DGlt) (244bp); and (3) small-target sequence (DGst) (152bp). After HRM analysis, three melting peaks are detected in male DNA samples and two in female DNA samples. An imbalance between the DGst and DGlt melting peak heights allows for the estimation of the extent of DNA degradation. For sensitivity assessment, triplicate aliquots of 0.0032 to 50ng/µL DNA were tested, denoting good linearity and reproducibility. The results also showed the analysis to be precise and accurate in the DNA range of 0.04-5ng/µL. Diverse types of DNA samples were tested: experimentally heat-degraded DNA; crime scene samples derived from casework and highly degraded samples with partial STR profiles from corpse material and mass disaster events. The results were compared with those obtained from the Plexor® and PowerQuant® commercial kits. Additionally, the quantification results of the QYDEG HRM triplex correlate well with the STR amplification that was subsequently obtained. The method is simple, cost-effective and helpful for determining the DNA integrity and the sex of a sample donor in any field where human DNA quantification is required.