Application Value Of Short Fragment Molecular Markers In Forensic Evidence Identification

An accurate identification of biomaterials at crime scene can provide strong evidence for case investigation and crime reconstruction.Identification of biomaterials at the species level and the successive tissue level are the two important kinds of forensic identification.Due to the particularity of forensic biomaterials,traditional forensic identification always has limits,calling for the molecular biological methods with high sensitivity,specificity and stability.Mitochondrial 12S rRNA and 16S rRNA gene have been used for species identification in forensic materials because of their multiple copies,intraspecific genetic stability and interspecific differences.Meanwhile,miRNA has been used for tissue identification in body fluid samples by merits of their stability and specificity.However,these genetic markers and their applied methods are always lack of universality and consistency in practical application.In this study,we developed the novel methods for species identification and body fluid identification,and provided evidences to support their feasibility.In the part of species identification,the mitochondrial 12S rRNA and 16S rRNA genes were selected and studied in this study as follows.First,the most variable regions of the sequences were identified through the sequence comparation across a broad range of mammal groups.Then the relatively conservative regions which close to both sides of the high variation region were selected to design short universal primers.Second,PCR amplification and sequencing analyses were carried out in some representative mammals of higher taxonomic rank.The secondary structure information and nucleic acid sequence information were combined to resolve species after the prediction and conversion coding of RNA secondary structure.Meanwhile,based on the conservative feature of the secondary structure,we evaluated its value in guiding homology alignment of distant species.Species identification using 12S rRNA and 16S rRNA short regions showed that:(1)The success rate of PCR amplification,sequencing and species identification in this study were all 100%.(2)Compared with the conventional alignment methods,secondary-structure guiding alignment improved the sequence alignment and phylogenetic relationship in higher taxonomic rank to a certain extent.(3)Secondary-structure information increased the additional genetic information for phylogenetic analysis:the genetic information,variable characters and parsimony-informative characters of 12S rRNA increased by 23.37%,20.69%and 20.86%respectively,and 16S rRNA increased by 17.95%,16.56%and 15.48%respectively.(4)Secondary-structure information increased the support value of some clades.In the 12S rRNA sequence-structure combination tree,about 20%of the clades had increased support value,ranging from 2%to 26%;in the 16S rRNA sequence-structure combination tree,13.64%clades had increased support value,ranging from 4%to 10%.(5)The species identification strategy based on secondary-structure information showed a certain potential in the identification of the race of human species.In the part of body fluid identification,an identification strategy was established for distinguishing blood type between menstrual blood and peripheral blood based on the copy ratio of miR-451a and miR-21-5p(451/21 Ratio).This strategy was tested and validated by using the standard curve method of approximate absolute quantification in 133 samples that covered five types of body fluids.The results showed that:(1)If 451/21 Ratio was greater than 0 and less than 0.929,the sample should be non-blood materials;if 451/21 Ratio was greater than 0.929 and less than 10.201,the sample should be menstrual blood;if 451/21 Ratio was greater than 10.201,it should be peripheral blood.(2)The accuracy of external validations in 62 menstrual blood samples and 24 peripheral blood samples was 100%,and this strategy was not affected by gender,age,period of menstrual blood,and exposure days.(3)The minimum dose of menstrual and peripheral blood for this strategy was 0.2 ng,and the repeatability among different experimental platforms and researchers was 100%.Moreover,this strategy was also suitable for the identification of menstrual blood with small number of mixed blood samples.(4)The relative stability of the 451/21 Ratio in menstrual blood agree with gene functions,wherein miR-451a and miR-21-5p might bond the target OSR1 to act on the endometrium and other reproductive organs or tissues,and then participated in the regulation of the normal development of the reproductive system.The new strategies of species identification and body fluid identification developed in this study are feasible and accurate,and combination of the two strategies can establish a comprehensive identification system from family and genus to tissue type,which is promising in forensic practice.