Problems of Forensic Sciences 2008 Vol. 76 (LXXVI) 355-368
HUMAN NUCLEAR AND MALE Y-CHROMOSOME DNA QUANTIFICATION BY REAL-TIME PCR ASSAY
Agnieszka MIROWSKA1, Ryszard PAWŁOWSKI2,3
1Faculty of Pharmacy, Medical University of Gdansk, Poland
2Institute of Forensic Medicine, Medical University of Gdansk, Poland
3Institute of Forensic Research, Krakow, Poland
Streszczenie
The aim of the investigations was to develop a novel method of simultaneous quantitative determination of human nuclear and male Y-chromosome DNA by real-time polymerase chain reaction (RT-PCR) assay for (the needs of) forensic genetics laboratories. When validating the method, the authors investigated the parameters and kinetics of both reactions. Combining two monoplex experiments in a single reaction was determined to exert no significant effect on the results and the investigators demonstrated a significant consistency between quantitative determinations of the two examined sequences. A very high sensitivity of the multiplex experiment was ensured by determination based on the AluYb8 sequences, thanks to which it was possible to detect with due accuracy at least 2 pg of human DNA. The described real-time PCR assay is sensitive to the degree of DNA fragmentation and for this reason it constitutes a prognostic tool that is helpful in selection of further DNA analytical methods. When evaluating the specificity of the reaction, the authors observed that animal DNA samples did not result in considerable amplification; nor did they interfere with human DNA determinations. Summing up, the developed new method of multiplex PCR assay not only provides reliable results of quantitative determinations of nuclear and male Y-chromosome DNA, but is also a source of information on the quality of the examined material.
Słowa kluczowe
Multiplex human DNA quantitation; Real-time PCR assay; Alu Yb8 sequences; Chromosome Y-specific sequence.