Problems of Forensic Sciences 2006 Vol. 68 (LXVIII) 351-361
DETERMINATION OF NUCLEAR DNA CONCENTRATION USING REAL-TIME PCR TECHNIQUE
Agnieszka MIROWSKA3, Ryszard PAWŁOWSKI1,2
1Institute of Forensic Medicine, Medical University, Gdańsk
2Institute of Forensic Research, Krakow
3Faculty of Pharmacy, Medical University, Gdańsk
Streszczenie
The general aim of the present study was to develop and optimise a real-time PCR (RT-PCR) based method of determination of human nuclear DNA concentration that could be utilized in forensic genetics laboratories. The study material consisted of human DNA as well as DNA from nineteen different vertebrate species. The developed quantitative test is based on amplification of the Alu Yb8 repetitive sequence specific for human DNA. It was shown that measurement of the nuclear DNA concentration is linear in the range from 200 ng/μl to 0.02 ng/μl and that appropriate precision and accuracy are ensured for samples which have DNA concentration above 2 pg/μl. The method should therefore be considered as very sensitive since it allows determination of DNA concentration at the level of the single haploid cell. A performed validation study indicated that in general amplification of animal DNA is negligible and it was also found that in the case of the (closely related to humans) chimpanzee species, an amplification signal of the Alu Yb8 is not present at all. The performed experiments showed that the developed method of determination of human nuclear DNA concentration is very sensitive, reliable and highly specific and therefore can be a valuable tool utilized in forensic examinations.
Słowa kluczowe
Forensic genetics; Determination of human DNA concentration; Real-time PCR; Alu Yb8 DNA sequences.