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Optimizing direct amplification of forensic commercial kits for STR determination
M Caputo1, M C Bobillo2, A Sala1
1Universidad de Buenos Aires, Facultad de Farmacia y Bioquímica, Departamento de Microbiología, Inmunología y Biotecnología, Cátedra de Genética Forense y Servicio de Huellas Digitales Genéticas, Junín 956 C1113AAD, Buenos Aires, Argentina; CONICET - Consejo Nacional de Investigaciones Científicas y Tecnológicas, C1033AAJ, Buenos Aires, Argentina.
This study presents an optimized workflow for direct DNA amplification from blood samples on various substrates, significantly reducing analysis time and costs in forensic genotyping. The streamlined process ensures high-quality DNA profiles with improved efficiency and lower failure rates.
Area of Science:
- Forensic Science
- Genetics
- Biotechnology
Background:
- Direct DNA amplification in forensic genotyping is crucial for reducing analysis time with large sample sets.
- Amplification success is influenced by PCR chemistry and the solid substrate used for sample deposition.
Purpose of the Study:
- To develop and optimize a time- and cost-effective workflow for direct DNA amplification from blood samples on diverse absorbent substrates.
- To evaluate the performance of a unified strategy involving pre-treatment, scaled-down PCR, and a specific analytical platform.
Main Methods:
- A total of 770 blood samples were analyzed from four different substrates: Blood cards, Whatman® 3MM paper, FTA™ Classic cards, and Whatman® Grade 1.
- A unified workflow included low-cost pre-treatment, PCR amplification volume scale-down, and analysis on the 3500 Genetic Analyzer.
- Three different commercial multiplex STR direct amplification kits were employed.
Main Results:
- Achieved over 94% high-quality DNA profiles, with a reduced re-injection rate (average 3.2%) and amplification failure rate (less than 5%).
- Demonstrated consistent performance across different commercial kits with an average peak height ratio of 0.91 and intra-locus balance values from 0.92 to 0.94.
- Comparison with previous results confirmed the efficiency and effectiveness of the proposed modifications.
Conclusions:
- The developed protocol is highly efficient, producing optimal quality DNA profiles for forensic applications.
- The strategy is both time- and cost-effective, making it suitable for large-scale forensic sample analysis.
- This optimized workflow enhances direct DNA amplification from various blood-spotted substrates.
