Multiple Amplification Of COX-112SrRNA And16SrRNA Genes In Mitochondria DNA For Species Identification

Objective:Species discrimination is an important step before individual identification in forensic science practice. Proceeding species identification for kinds of biomaterial from scenes is the first and key link before individual identification. By taking advantage of multiple copies of mitochondrial DNA in single cell we established a high specific, sensitive,stable,reliable and handy multiplex amplification system for species identification which was adapt to practice and would solve problems and shortcomings in species identification at the present.Methods:Before exploring a method of multiple amplification-AGE for species identification. we consulted papers about species identification for reference and confirmed screening principles of human specific gene and reference genes. According to mt DNA sequence of human and other mammals in gene bank we screened eligible encoding genes and compared the sequence with the other mammals carefully. Then we found differences between human being and other species and figured out the target gene sequence. The primers were designed and optimized by software primer5and primer3(online) according to primer design principles. Many muscle or blood samples were collected including rhesus,pig, cattle, sheep and rabbits and human being. To get the best12SrRNA,16SrRNA and COX1multiplex amplification conditions we proceed the try-and-error strategy. DNA from different samples were extracted and amplified by PCR. The PCR products were separated by3%agarose gel electrophoresis. The bands were visualized under254nm UV lamp.Results:COX-1gene was chosen as human specific gene segment and other two genes12SrRNA and16SrRNA as internal control. Each gene was detected by one pair of primer except12SrRNA. We designed two pairs of primers for12SrRNA gene and these two PCR products share the same size. The result of agarose gel electrophoresis(AGE)showed that human sample had three bands, representing16SrRNA,12SrRNA and COX-1gene, with the lengths of395bp,231bp and202bp respectively. The other species including rhesus monkey showed only two bands,16SrRNA and12SrRNA (by12S1amplification). For samples from pig and cattle12SrRNA gene were amplified by12S2primer. Based on our observation the multiple amplification system’s sensitivity was as low as2.5pg. Also we can detect the human specific COX-1gene when10-8pg template was used. Therefore, we believe the multiple amplification system can be used in forensic casework test.Conclusion:This research initially established a multiplex amplification system for species identification in forensic science application. For routine samples, the human being can be recognized by our multiple amplification system. It seems that the system was reliable and sensitive enough for daily forensic science casework.