Statistical analysis of biological samples collection and DNA test results in 1574 larceny cases / 中国法医学杂志

Objective To analyze the 2496 biological samples collected from 1574 larceny cases and improve the application of DNA in the larceny cases. Methods Make a summary of the biological samples collection methods and DNA test results in different type of larceny cases. Results Touch samples have already become the most common type of biological samples in larceny cases, but their DNA test success rate are still low, more work should be done for the DNA mixed type to improve DNA hit. The DNA positive result rate had statistical difference in biological samples with different collection methods. For the Touch samples, flocked swab and original are the best collection methods. Conclusion More Valuable biological samples collected by the crime scene investigators are the Key factor in improvement of DNA detection capability, awareness of trace biological evidence and the touch sample collection technique are very important for the crime scene investigators.

High-resolution melting analysis in forensic research / 中国法医学杂志

High-resolution melting (HRM) analysis is a versatile method for variant scanning and genotyping. It involves amplification of the target in the presence of a saturation dye by the polymerase chain reaction (PCR). HRM analysis can be performed in one closed-tube, which does not require additional post-PCR separations and greatly reduces the possibility of contamination. HRM is faster, simpler, and less expensive than alternative approaches requiring labeled probes. Considering the many advantages of HRM analysis, many researchers have tried to apply this method to forensic research. This paper intends to summarize the principle, technical characteristics, limitations and application of HRM analysis in forensic science.

The influence of sample components on "Coffee Ring" DNA test / 中国法医学杂志

Objective To investigate the influences of drop components on the"coffee ring" test of DNA. Methods The DNA-EB drops containing SDS, NaCl, etc, were evaporated on glass slides. After evaporation, the fluorescent Deposition Pattern (DP) and Integrated Optical Density (IOD) was acquired with a gel imaging system. The dose-effect relationship was analyzed. Result When the non-DNA components concentration is low, the DP was still characteristiced by peripheral rings, subtle component-specific differences, such as tree-ring like structure and radial crack, existed. At high concentrations, DP was various, which may be ring + scattered dots (NaCl), central spot + small weak ring (SDS), concentric/tree-ring (TritonX-100) or ring + spot (H+), et al. Non-DNA components had little effect on IOD(0.5~2 times). Conclusion Non-DNA components in DNA-EB drops influences both the DP and IOD, but rough estimation of DNA concentrations is still effective. Moreover, analyzing the DP can provide more informations about sample purity and residue.

Research on DNA mixture de-convolution with stochastic simulation method / 中国法医学杂志

Objective To analyze a large number of DNA mixture de-convolution data with stochastic simulation method. Methods Using the software in Identifiler, PP18D, AGCU EX20, PP21, AGCU EX20 & AGCU 21+1 system, 1 million groups of STR genotyping of mixture were analyzed. The average de-convoluted loci (L), the average de-convoluted combinatorial number (Π) were counted out. Results In identifiler, PP18D, AGCU EX20, PP21, AGCU EX20 & AGCU 21+1 system, L were 4.8, 5.8, 6.7, 7.0, 11.1, and Π were 3.71×104, 1.06×105, 3.34×105, 6.40×105, 2.48×1012. Conclusion The result in this paper has some guiding significance in DNA mixture de-convolution followed by DNA Database search.

The identification of the 16SrDNA of the diatoms in rabbits' internal organs in the summer and winter in Yongjiang River of Ningbo / 中国法医学杂志

Objective To evaluate the application value of identification on drown by detection 16SrDNA of the diatoms in rabbits' internal organs in summer month of July and winter month of December in YongJiang River of Ningbo. Methods 60 Rabbits were randomly and medially divided into three groups in summer and winter: drowning group, postmortem immersion group and using only lethal aeroembolism as control group. Specimen including heart, liver, lung and kidney from each rabbit were tested with diatom 16SrDNA PCR method. Results Compared with postmortem immersion group, detection rate of diatom 16SrDNA of heart, liver, lung, renal tissue in drowning group was significantly higher than that in summer month of July (P<0.05), In December, the 16SrDNA of the drowning group was detected in heart and lung tissues, There was no significant difference compared with postmortem immersion group (P>0.05) In summer month of July, detection rate of 16SrDNA of heart, liver, lung, renal tissues in drowning group was significantly higher than that in winter month of December (P<0.05). Diatom 16SrDNA of heart, liver, lung, kidney tissues in air embolism group were not detected In summer month of July and winter month of December. Conclusion With the higher detection rate of diatom 16SrDNA in drowning rabbit in summer, the diatom 16SrDNA PCR method can be used for the diagnosis of drowning in Yongjiang River of Ningbo; while in winter , it should be carefully apllied with the lower detection rate of diatom 16SrDNA.

Comprehensive application of multiple methods in determination of half-sibling relationship / 中国法医学杂志

Objective To identify the half-sibling relationship by comprehensive using three different methods. Methods STR genotype was performed on A, A's mother, B and B's mother by using PowerPlex21 kit, AGCU Expressmarker 21+1 kit, Microreader 23sp-B kit and AGCU X-19 STR kit respectively. Based on the results of STR genotype and X-STR, we determined half-sibling relationship by ITO, discriminant function analysis and IBS. Results HIS was between 1.36×102and 2.09×105in ITO which indicated that A and B had the same father. The IBS and discriminant function analysis also had the same conclusion. Conclusion Comprehensive using multiple methods can obtain reliable result to identify the half-sibling relationship from the same father.

An exploratory study of effectiveness with ethylene oxide treatment for removing DNA contamination / 中国法医学杂志

Objective This exploratory study aimed to assess effectiveness with ethylene oxide treatment for removing DNA contamination. Methods 98 different spiked samples such as saliva, dander, skin cell, hair, blood and cartilage were conducted with ethylene oxide treatment. After extraction of samples, the dna was amplified and then the STR analysis was performed with 3130xl or 3500xl. Results A 6h EO treatment results showed that two saliva stains of 44 samples STR profile were detected; Just one hair of 54 samples treated with ethylene oxide was detected contaminating DNA with EO treatment for 8 hours. Conclusion This work suggested that it was more successful to reduce DNA contamination by using ethylene oxide treatment.

Advances in the research of predicting the time since deposition of evidentiary stains / 中国法医学杂志

In addition to obtaining DNA-STR typing of an evidentiary stain for individual identification and paternity tests, knowing the time since deposition (TSD) is also highly desired in forensics. To provide a reference for the research of predicting the TSD, this article reviews the reported optical, cell biological and molecular biological methods of determining the age of bloodstains domestic and overseas, and also introduces the application of microbial forensics, a new field of forensic science, to provide space-time clues of evidentiary stains.

The study on DNA mixtures / 中国法医学杂志

DNA mixture has been a problem to be conquered for a long time in the forensic study. The DNA mixtures can be mainly divided into two categories: one comes from the sex assaults, for which we have to detect the sperms among the large amount of vaginal epithelial cells; the other one is the combination of different common samples, such as blood mixtures, salvia-blood mixtures and so on. In recent years, deeper and deeper study on DNA mixtures has led a way to objectiveness and pluralism. Novel techniques, like fluorescence- or magnetic-activated cell sorting strategies,micromanipulation and acoustic different analysis can be utilized to separate sperms in the mixtures; as for the second sort, the application of massively parallel sequencing(MPS), microfluidic droplet, whole-genome amplification, emulsion PCR(ePCR) et al. rise up the chances to detect the minor contributor in the mixtures. Furthermore, the development of both traditional and novel biomarkers which includes STR, Y-STR, SNP, DIP, Microhaplotype, DIP-STR, SNP-STR,, mtDNA-SNP and so on, provide us more analyzing choices in mixture study. The last part of the assay focuses on the latest progresses of evaluating the number of contributors, the explanation theory and calculation software.

Technical feasibility study on the application of circRNA in body fluid identification / 中国法医学杂志

Objective Using circRNA detection technology to explore the feasibility of the application of circRNA molecules in the identification of body fluids. Methods Prepare three kinds of body fluid samples: semen, saliva and vaginal secretions. Total RNA was extracted from Qiagen RNeasy Micro kit and digested by RNase R to obtain circRNA. Reverse transcription PCR amplification and agarose gel electrophoresis analysis were performed to detect target products. Results CircRNA can be detected in all prepared samples. These results showed that the circRNA was widely present in common body fluids of forensic medicine, and had some application value. Conclusion The detection for circRNA can be compatible with the existing DNA detection technology, and its tissue specificity can be used as a new marker for identification of body fluid and has important research value.

Next generation sequencing-based precision STR genotyping of mixed samples / 中国法医学杂志

Objective To genotype mixed samples with next generation sequencing and evaluate its prospects in forensic DNA application. Methods Three mixed biological samples from rapes cases and their reference samples were collected. DNA was extracted using the MagAttract M48 DNA Manual Kit(200). The ForenSeqTMDNA Signature Prep Kit was used for library preparation, and next generation sequencing was performed on the MiSeq FGx system. The ForenSeqTMUniversal Analysis v1.2.1 software was used for data analysis. NGS-based STR results were compared with CE-based genotypes. Results A single length polymorphic STR allele in the mixed profile could be recognized as two sequence polymorphic STR alleles from different donors, which would assist mixed profile analysis. Such phenomenon was observed in D3S1358, D9S1122 and D13S317 in this work. Conclusion Our results suggested that precision STR genotyping of mixed samples based on NGS can provide more information and hints for mixed STR profile separation.

The application of a high-performance bloodstain testing reagent in forensic scene / 中国法医学杂志

Objective To develop a highly sensitive luminescent reagent for bloodstain testing at forensic crime scenes.Methods Based ontheprincipleof ECL luminescence and the ping-pong conjugate activation principle of chemical electronic chain,this project developed a new type of highly sensitive luminescent reagent for bloodstain testing by usingthe uniform design of experimental methods to optimize the conditions andsynthesize several new compounds.Results The bloodstain testing luminescent reagentdeveloped in this project has high sensitivity andlongluminescence time.In the case of blood samples diluted by 1,000 times,reading the fluorescence withChemiScope 3300 chemiluminescence imaging system,the maximumvalue of gray scale reached 56,and the luminescence time lasted for 10 minutes.Conclusion The project has successfully developed a highly semitivebloodstain testing reagentthat could be applied to crime scene investigation.

Typing of fragment length polymorphism marker with primer SNP-binding at the last second 3'end / 中国法医学杂志

Objective To successfully get the full PCR alleles of the Insertion/Deletion marker rs10644346 in which a SNP-binding exists at the 3'end region of the primer.Methods Based on the AS-PCR,a common forward primer and two reverse primers with allele-specific oligonucleotides at the last second 3'end instead of the terminus were tentatively designed for typing 150 unrelated individuals and 10 father-mother--child trios from Htnan province in South-central China.Simultaneously,9 samples were typed with all the above three primers (the two primer sets which consist of the common forward primer and one of the reverse primers).Results PCR amplicons were well detected in the 150 unrelated individuals after being typed with the three primers,and the amplified fragments of parental and filial generations among 10 father-mother-child trios conformed to Mendel's principles.Allele missing was found in the two-primer group.Conclusion The primers designed by locating the specific nucleotide at the last second 3'end rather than terminal position were demonstrated also effective in getting specific alleles if perfect mismatch and PCR conditions are guaranteed,and the design strategy can provide an optional reference to rescue markers of SNP-binding primers for forensic practice.

Advances in the research of genome-wide association studies on healthy adult height and missing heritability / 中国法医学杂志

Human height, a complex trait with over 80% heritability, is determined by genetic and environment factors. Currently, a certain amount of specific variants about human height have been found, especially with the widely used of genome-wide association studies (GWAS) in genetics research, making it a great progress for the discovering of height associated genes. However, single nucleotide polymorphisms (SNPs) found by GWAS can only explain a minority of the heritable. This article reviews the progress of GWAS about height associated genes and missing heritability domestic and overseas.

Comparative?analysis?of?two?different?DNA?purification?methods?for?bones?and?teeth / 中国法医学杂志

Objective To compare the effect of silica-extraction method and Silico membrane based method in DNA purification from bones and teeth.Methods DNA samples were purified respectively with the silica-extraction method and MinElute PCR Purification kit from 6 bones and 8 teeth,then tested STR types by GlobalFiler? kits. And evaluated the two methods with the success rate and the peak height. Results Both of the two purification methods can successfully obtain the STR markers of the 14 samples. And there was no statistical difference between the two methods in the average peak height from bones and teeth. Conclusion The Silico membrane based method which have more advantages in operation is an efficient method to purify DNA from bones and teeth, and there is no significant difference compared with the silica-extraction method. But the cost is higher. It can be selectively used in forensic practice.

The pilot study of the phasing method for STR haplotypes / 中国法医学杂志

Referring to the statistical model for the SNP haplotype phasing which was based on the allele frequencies and advised by Browning SR, we investigated and deduced the phasing method of STR haplotype with linkage disequilibrium inpaternity testing preliminarily. Haplotype phasing of two X-STRs in linkage disequilibrium were illustrated. This method provides an idea for the haplotype phasing of STR markers, which is helpful for interpreting the typing results of STR more scientifically and accurately.

The study for the DNA case work sample purification by the automated DNA extraction system with magnetic beads / 中国法医学杂志

Objective To evaluate the forensic application of TE-MAGS technology based on magnetic beads kit on TECAN pipetting platform and establish the automated DNA extraction system of case work samples. Methods Sensitivity test: 10 different DNA samples from 0.1ng to 1ng were prepared with a commercial standard DNA 9947A diluted into 200μL TES. DNA samples were purified by the TE-MAGS technology automatically on the TECAN pipetting platform and then typed using the IdentifilerTM Kit and get the profile of STR with the software GeneMapper ID-X; the power of purification was tested with a trail that purified 1ng DNA mixed with humus acid and hemachrome. Comparative test: 304 casework samples were divided into two purified by TE-MAGS technology and silicon beads respectively to compare the power of purification and the possibility of forensic utility. Results Sensitivity test: 0.3ng and more imported DNA can obtain a good quality of DNA profile compared to the lower imported DNA with dropout of STR peaks (0.1ng and 0.2ng). The power of purification of the TE-MAGS technology was not affected by humus acid and hemachrome. The comparison result between automatic TE-MAGS technology and manual silicon beads extraction methods from 304 casework samples showed that the former's success rate(50%) was higher than the latter(40.8%). Conclusion The established DNA purification method of TE-MAGS technology automatic DNA extraction system in this study was obviously advantaging at the aspect of success rate, stability, and uniformity and suited to application in the forensic utility future.

Analysis of mutation of 20 autosomal short tandem repeat loci in Henan Han population / 中国法医学杂志

Objective To investigate the mutation phenomenon of 20 autosomal STR loci in Henan Han population. Methods A total of 3011 parentage confirmed cases were collected to screen mutation events, ascertain the source of mutation, calculate mutation rate, analyze mutation rules and compare with the mutation condition of populations in different regions. Results 76 mutation events were observed in 19 STR loci, the average and accumulative mutation rate reached to 0.08% and 1.6629%, respectively. The ration of paternal versus maternal mutation was 8:1. Mutation rates of Penta E and D12S391 loci in Henan Han population were lower than the Han population of northern China(P<0.05); the mutation rate of CSF1PO locus were lower than Guangdong population and Yunan Han population(P<0.05); the mutation rates of D6S1043 and D12S391 loci were lower than Guangdong population(P<0.05). Conclusion STR mutation events were common in paternity testing. Region differences among mutation rate were significant.

Application of next-generation sequencing technology in forensic science / 中国法医学杂志

With the rapid development of Next-generation Sequencing(NGS)technologies, and its high throughput and low cost is applied widely in the field of life science, the increase in the depth of sequencing together decrease in the consumption of time and cost, makes a wide application of NGS in the research of microbiological research, ancient DNA study, clinical diagnosis, forensic science research, etc. The article discusses the second generation sequencing technology platform and its genetic markers in the forensic application. Included STR typing, SNP typing, HLA genotype prediction and the application in the degradation of the material.

Identify normal and abnormal semen via mRNA fluorescence multiplex amplification system: A preliminary study / 中国法医学杂志

Objective Construct a mRNA multiplex amplification system to identify different types of semen stains. Methods First, collect normal, oligozoospermia and azoospermia semen samples to make semen stains. Second, extract total RNA with Qiagen RNeasy Micro Kit. Then use reverse transcript PCR to amplify goal mRNA markers: 2 markers for sperm(PRM1, PRM2), 2 markers for seminal plasma(TGM4, SEMG1) and 2 housekeeping genes(TEF, UCE). Results All semen mRNA markers can be detected in normal semen samples. The RFU of sperm mRNA markers are lower in oligozoospermia semen samples than that in normal controls. No sperm mRNA markers can be detected in the azoospermia semen samples, only seminal plasma specific can be detected. Conclusion The differentiation of normal and azoospermia semen can be achieved by using multiplex mRNA fluorescence amplification system. While normal semen and oligozoospermia semen compared to no statistical difference.