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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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DNA typing of biological samples is an important step in performing individual identification and paternity testing in forensic applications. In practice, the detection of complex biological samples and the identification of complex kinships are challenging current biological technologies. Novel DNA technologies are also introduced into forensic genetics to improve the power of analysis. Next-generation sequencing (NGS) has several advantages, such as high-throughput and low cost, and can obtain detailed DNA sequences and relative contents of targeted regions, which will improve the detection of biological samples to help the analysis of forensic cases. The application of NGS in forensic genetics has received extensive attention and reports on the application of NGS in forensic genetics are increasing. In this study, we summarized the progress in the application research of NGS in the forensic genetics including the detection of genetic markers and their analytical methods. This will provide guides for related studies and forensic applications.
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Recently introduced microhaplotype has attracted more attention in forensic field. A microhaplotype is a short segment of DNA (e.g<200bp) contains two or more SNPs. The microhaplotypes showed extremely low mutation rates. The amplicons of alleles are balance to each other and without any stutters. By these advantages, it shows great value for forensic application, such as mixture stains analyzing, ancestor analysis, and kinship analysis. Thus, we aimed to review the development, analytical approach, nomenclature and population characteristic of microhaplotype.
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Objectives To conduct a research on the possibility and effect factors of latent fingerprints development in clothing objects after vacuum coating, and extracting fingerprints DNA and to probe in the relation among DNA template quantity and genetic loci numbers tested, and the rfu value after coating. Methods To select two groups that are free sweat hands and sweat hands and have them press their fingerprints on the cloth, after coating, and to analyze the effect of time, to quantify and test the targeted fingerprints DNA, to compare the locus numbers tested between white and black cloth. Results As the time is prolonged, the locus numbers tested decrease. The locus numbers tested on the group of sweat hands using the same method after the same placed time are lager than the free sweat hands. When the value of rfu is 600 above, the ratio of the locus numbers tested is more than 90% and the threshold of templates is 0.013ng. The locus numbers tested of white cloth is larger, comparing with black cloth when using the same method. What is more, there exists an prohibitive influence of pigments of the dyed cloth over the PCR amplification, to put it further, the loci numbers tested will be trimmed. Conclusion The technology of vacuum coating can be well used in the area of detecting fingerprint DNA.
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Objective To investigate genetic polymorphism of OSU49 locus on Y chromosome among 300 unrelated males in Henan Han population and to assess its value in forensic science. Methods Under the case of informed consent, the blood samples were collected from 300 male individuals in Henan Han. The primer was labeled with florescent. PCR products were separated from ABI 3130 genetic analyzer. According to the typing test results, the sequence of different alleles was analyzed. Result OSU49 locus contained both pentanucleotide and tetranucleotide core sequence. In the Han population of Henan, the motif of OSU49 locus was showed (CTTTC)pCTT(CCCT)7 T(CTTTC)1(TCTT)5(TCCT)m(TCTT)nTCT(TCCT)4. The number of pentanucleotide repeats varied from12 to 17, and the number of tetranucleotide repeats varied from 20 to 30. Nomenclature for allele was according to the length of the fragment. A total of 34 alleles were detected. The gene diversity was 0.9186 and the discrimination power was 0.9155. Conclusion The complex repeats of locus OSU49 was highly polymorphic in Henan Han population, which can be used in forensic science and human genetics studies.
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Objective To explore the mutation types and disciplines of STR commonly used in forensic in gynecologic and breast cancerand investigate the application of microdissection in forensic practice involving tumor tissue. Methods DNA of tumor tissues, adjacent normal tissues and peripheral blood from 62 patients with breast cancer, 62 patients with gynecologic cancer and 10 patients with benign gynecologic tumor were amplified by PowerPlex 21 System kit and Argus X-12 kit. Capillary electrophoresis of PCR products was carried out on an ABI 3130 Genetic Analyzer to obtain genotypes. Some tumor tissues with STR variation were microdissected. Results The genotype of peripheral blood in cancer patient was consistent with that of corresponding normal tissue. 4 types of STR variations were found in 46.77% gynecologic cancer tissues, compared with that in benign tumor tissues and breast cancer, the difference of STR variation was significant(P<0.01,P=0.009). The genotype of stromal cells separated by microdissection was consistent with that of corresponding adjacent normal tissue. Conclusion The STR loci detected in the study with poor stability are not suitable for forensic cases involving gynecologic cancer tissues. The genotype of stromal cells separated accurately from tumor tissues by microdissection could represent the normal DNA genotype of the individual with cancer. Microdissection is an effective solution in forensic cases with tumor tissues.
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Objective with the advantages of rapidity in detection protein, We selected the gender-specific amino acid sequence based on human SMCY and SMCX, cloned and expressed SMCY gender-specific fusion antigens. The rabbits were immunized with purified antigens to obtain the polyclonal antibodies. A new method was established for rapidly sex identification of forensic evidence samples by detecting SMCY antigens with the corresponding polyclonal antibodies. Methods We found three differential fragments by analyzing the sequence of human SMCY and SMCX. Then we cloned this three fragments and ligated as a new recombinant.This SMCY gender-specific fusion antigen gene was sub-cloned into pET-28a and expressed in Escherichia coli. The fusion antigen was purified by Ni-NTA column. The rabbits were immunized with purified antigen to produce the specific polyclonal antibodies.The reactivity of the polyclonal antibody was evaluated by ELISA and Western blotting. We developed a colloidal gold test strip for detecting the gender of human samples. Results We successfully selected gender-specific amino acid sequence, the SMCY gender-specific fusion antigen was expressed by prokaryotic expression and the polyclonal antibody was prepared by immunizing rabbit. The results of colloidal gold strip tests showed that there is a significant difference between male and female serums. Conclusion The results showed that the SMCY gender-specific fusion antigen could be recognized by the polyclonal antibody.The colloidal gold strip tests made by SMCY gender-specific fusion antigens and the corresponding polyclonal antibodies could be used for rapidly determining the gender of forensic evidence samples.
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Objective To compare the PCR effect of rapid PCR instrument and common PCR instrument. Methods The concentration of 9947A standard was diluted to 0.1, 0.05, 0.025, 0.0125, 0.0063 ng/μL, 100 samples from each concentration group to establish PCR reaction system with Identifiler? Plus PCR Amplification Kit, 50 samples of them test with rapid PCR instrument (Speed cycler2 thermocycler), the other 50 samples test with common PCR instrument (9700 thermocycler). Detection of PCR product with 3500xL Genetic Analyser, the STR typing of both groups of each concentration group should be compared. Results The success rate of both thermocyclers have no significant difference (P>0.05); The success number of STR typing of common PCR instrument (13.7±1.0; 11.3±1.5) were higher than rapid PCR instrument (13.1±1.3; 9.9±1.9) when the concentration of 9947A were 0.0125, 0.0063ng/μL (P=0.029; P<0.001); The peak height of DNA typing map obtained from common PCR instrument (18931±4625;13437±3165; 5752±1344) were higher than rapid PCR instrument (16929±4034; 11815±4120; 4865±1401) when the concentration of 9947A were 0.1, 0.05, 0.025ng/μL (P=0.023; P=0.030; P=0.002). Conclusions The rapid PCR instrument could achieve the equal success rate to the common PCR instrument with less time, which revealed that the rapid PCR instrument was suitable for application in practical cases; The quality of STR typing from common PCR instrument may be more higher.
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Objective A new methodology was established to efficiently obtain the genotype of DNA remained on standard long gun. Methods Direct PCR and silicon membrane method were combined to detect DNA polymorphism of a total of 240 samples at 5 different positions from 48 standard long guns. Results Combining direct PCR and silicon membrane method, we obtained full DNA profiles in 42 out of 48 standard long guns, with a detection rate up to 87.50%. Conclusion The results demonstrate that the combination of direct PCR and silicon membrane method provide a quick and accurate way to detect DNA polymorphism on the standard long gun.