Establishment Of Saliva-specific MRNA Polymorphism Detection System And Its Exploration In Forensic Mixture Analysis

Objective:Using polymorphic SNP markers in saliva-specific mRNA molecule,we establish a detection system which can identify the provider of saliva component and indicate the kinds of body fluids in the forensic mixture.Methods:Combined with multiplex PCR technology and SNaPshot minisequencing assay,the establishment of detection system which can get the typing result of 12 SNPs from 4 saliva-specific mRNA molecules in forensic mixture,and indicate the tissues/body fluids types that may be contained in forensic mixture by other tissues/body fluids indicator markers used specific expression of mRNA molecules in different tissues/body fluids.Following criteria about the selection of specific mRNA molecules in different tissues/body fluids and SNPs in saliva-specific mRNA molecules,and the PCR primers design has been accepted:(1)it is widely accepted that 4 saliva-specific mRNA molecules and 5 other tissues/body fluids specific mRNA molecule markers have been reported in literatures;(2)mRNA molecular markers are abundantly expressed in specific tissues/body fluids,and there is no expression or only a small expression in other body fluids;(3)the minor allele frequency(MAF)of SNPs in saliva-specific mRNA molecules is greater than 0.1;(4)the SNPs should be located within 300 bp of exon-intron junction,in order to design primers across different exons,ensuring that PCR amplification are not affected by DNA;(5)all amplicons are less than 300 bp,which makes the detection system more suitable for the analysis of forensic degradation sample.After RNA extraction and reverse transcription,multiplex PCR amplification is performed to obtain the amplicons.Then SNaPshot minisequencing kit is used for single base extension(SBE)and capillary electrophoresis(CE)detection to get the typing results of the 12 saliva-specific SNPs and other five possible tissues/body fluids indicator markers,to identify the saliva component in mixture and speculate the composition of the mixture.Results:1.We have successfully established a SNaPshot minisequencing detection system containing 12 saliva-specific SNPs and 5 common tissues/body fluids(peripheral blood,menstrual blood,semen,skin and vaginal secretion).Meanwhile,we have evaluated the specificity,sensitivity and aged sample detection capability of our system,showing well.2.After all home-made mixtures were successfully tested,we obtained the typing results of saliva components and estimated composition of mixtures correctly,including mixtures of saliva-peripheral blood and saliva-menstrual blood mixed with the ratio of 1:50;saliva-semen,saliva-skin,saliva-vaginal secretion mixed with the ratio of 1: 100;saliva,peripheral blood,menstrual blood,semen,skin,vaginal secretion from 6 contributors the minor RNA of saliva mixed with the ratio of 1:50.3.The typing results of 12 SNPs in saliva-specific mRNA were obtained by our detection system and Sanger sequencing,showing the same results with database.4.By typing the saliva samples of 100 healthy unrelated Chinese Han individuals residing in Shanxi,we evaluated the combine discrimination power(CDP)of this detection system.Conclusion:The saliva-specific SNP typing detection system was established by using SNP polymorphism markers in saliva-specific mRNAs and other five common body fluid indicator markers with the SNaPshot minisequencing assay.It provides a new method to identify the saliva components in forensic mixture.In the practical application of forensic science especially in sexual assault cases,it is of great significance to analyze the mixtures that may contain saliva component and can provide more comprehensive scientific evidence for case detection.