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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 To prepare the standard molecular weight fragment mixtures. Methods Primers were designed to prepare clones which contained different sizes of standard molecular weight fragments. The template used for amplification of insert fragments was the pMD18-T vector. Bacteria culture and plasmid extraction were used to obtain abundant target fragment. Unlabeled DNA fragments were prepared by double digestion of the recombinant plasmids, and the fluorescent adaptor was prepared by annealing with two partial reverse complimentary DNA fragments. The unlabeled fragments and fluorescent adaptor were connected by DNA ligation reaction assisted with T4 DNA ligase. In this way, different sizes of standard molecular weight fragments were prepared. Standard molecular weight fragment mixture was finally prepared by mixing all the fragments together before purification. Results Ten standard molecular weight fragments of different sizes were prepared. The sizes of each fragment are 80bp, 124bp, 194bp, 224bp, 254bp, 304bp, 349bp, 399bp, 424bp and 454bp. The internal standard could accurately determine the size of PCR products amplified with the DNATyper15 kit. Conclusion Using this method, the standard molecular weight fragment mixture which meet the requirements of research and laboratory use was prepared, perfectly providing a new method for preparation of the DNA molecular weight standards. The peaks and the size of the prepared DNA internal lane standard are correct, which can be used to calculate the DNA fragments size in capillary electrophoresis.
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Objective To discuss the risk in duo paternity testing. Methods 22 fictitious duo families formed by 22 pairs of unrelated individuals having one or zero inconsistent locus were selected after detected by GoldeneyeTM 20A Kit. The 22 fictitious duo families were further tested with STRtyper-10G kit and/or AGCU 21+1 STR kit until there were more than 3 inconsistent loci and the cumulative paternity index(CPI) value was less than 0.0001. According to the three excluding rules, ① number of inconsistent loci>3; ② CPI ≤ 0.0001; ③ accord with both ① and ② , using multiple STR systems, such as 19 STR loci, 26 STR loci, 39 STR loci and 46 STR loci to test and discuss whether there is difference among the excluding result of unrelated individual. Results Among those 22 fictitious duo families, using three excluding rules, None was excluded by 19 STR loci, and all was excluded by 39 STR loci. Conclusion Duo paternity tests may get a wrong result using only 19 loci system. To reduce the error risk 39 STR-loci systems would be suggested.
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Objective To construct a database for the genetic polymorphism of 19 STR loci in Han population from Hainan province. To investigate the application of 19 STR loci in the paternity testing. Methods The genotypes of 462 unrelated individuals in Hainan were detected with GoldeneyeTM 20A PCR Amplification Kit. 19-STR database was acquired, analyzed and evaluated in 283 paternity testing cases. Results No deviations of allele frequency from Hardy-Weinberg equilibrium expectations were found for Chi-square test (P>0.05). Observed heterozygosity (Hobs) varied between 0.603 and 0.914, total discrimination power (TDP) of 19 STR loci was more than 0.999999999999999, cumulative probability of exclusion (CPE) for triplet cases was 0.999999994. In all 283 paternity testing cases, triplets and duos were 170 and 113 respectively; there were 36 (12.7%) excluded cases comparing to 247 confirmed cases (87.3%). 14 mutation events were observed, and all were one-step mutation. Conclusion 14 out of 19 loci showed highly polymorphic in Han population from Hainan, and 19 STR system has high cumulative probability of exclusion and can meet the needs of paternity test of the local region. But mutation should be paid special attention to.
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As is known, H-Y antigens (male specific minor histompatibility antigens) are a group of minor histocompatibility antigens encoded on the Y-chromosome with homologous H-X antigens on the X-chromosome. H-Y antigens were originally discovered as transplant antigens, and they are only expressed in male individuals without specificity for different tissues and organs. A lot of research results show that H-Y antigens play an important role in the sex selection and identification. The paper reviews the above and discusses the application prospect of H-Y antigen in forensic science.
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Background and purpose: Short tandem repeats (STR) multiplex PCR fluorescence detection technology is the most widely used DNA technology in individual identity and genetic identification. It's the most direct method to obtain accurate conclusions. However, some studies have indicated that the rate of STR mutations in tumor tissue is significantly higher than that in normal tissues or blood. This study aimed to investigate the tendency of genetic instability in 20 STR loci on autosomal and Amel loci in tumor tissue samples from lung cancer. Methods: This study, collected 75 cases of human lung cancer tissues and the adjacent normal tissues. DNA samples were extracted by tissue DNA extraction kit, amplified using MicroreaderTM 21 Direct ID System PCR amplification kit. Capillary electrophoresis was performed using API 3130 analyzer, and results were analyzed by genetic analysis software (Gene Mapper ID V3.2). Results: STR alterations were detected in 24 specimens from 75 lung cancer tissues (32%). Fifty-five alterations were detected in the frequently used 21 STR loci in total, including additional alleles 10 times, loss of heterozygosity 10 times, partial loss of heterozygosity 35 times. Partial loss of heterozygosity was the most common genetic alteration types accounting for 63.64% of the total alteration frequency. And multiple genetic alteration types could occur in the same lung cancer tissue. Among them, the highest alteration frequency occurred on D5S818 (7 times), secondly on D3S1358 and D12S391 (both 5 times), and no alterations on D2S441 and Penta E. Combining the experimental results and analysis on clinical data, this study found the statistical differences between the staging of lung cancer and the age of the patients with the STR loci alterations (P0.05). Conclusion: STR loci of the lung cancer tissue were not stable, and the alteration occurred in the aged or high malignant degree lung cancer tissue more frequently. Meanwhile, no alteration was detected on D2S441 and Penta E. In the future research the two STR loci should be verified to determine whether they can be used as the stable STR loci in such cases by increasing the sample size.
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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.