Rapid analyzing mixed STR profiles based on the global minimum residual method.

The analysis of mixed short tandem repeat (STR) profiles has been long considered as a difficult challenge in the forensic DNA analysis. In the context of China, the current approach to analyze mixed STR profiles depends mostly on forensic manual method. However, besides the inefficiency, this technique is also susceptible to subjective biases in interpreting analysis results, which can hardly meet up with the growing demand for STR profiles analysis. In response, this study introduces an innovative method known as the global minimum residual method, which not only predicts the proportion of each contributor within a mixture, but also delivers accurate analysis results. The global minimum residual method first gives new definitions to the mixture proportion, then optimizes the allele model. After that, it comprehensively considers all loci present in the STR profile, accumulates and sums the residual values of each locus and selects the mixture proportion with the minimum accumulative sum as the inference result. Furthermore, the grey wolf optimizer is also employed to expedite the search for the optimal value. Notably, for two-person STR profiles, the high accuracy and remarkable efficiency of the global minimum residual method can bring convenience to realize extensive STR profile analysis. The optimization scheme established in this research has exhibited exceptional outcomes in practical applications, boasting significant utility and offering an innovative avenue in the realm of mixed STR profile analysis.

Research in forensic medicine under the view of biosafety.

In recent years, biotechnology is gradually getting popular and is playing a significant role in human productivity and life. The consequent biosafety problems are becoming increasingly prominent. Based on the connotation and extension of biosafety, this article sorts out the biosafety contents involved in traditional and modern forensic medicine research and analyzes the risks and challenges facing forensic medicine research from the perspective of biosafety. Based on the protection of legal medical experts, the establishment of working standards, and the promotion and support of research in forensic medicine on biosafety field and other aspects, this article discusses the prospectives of forensic medicine research from a biosafety point of view, and provides the insights and references for a smooth implementation of forensic medicine practice in the future.

Developmental validation of the DNATyper™Y26 PCR amplification kit: An enhanced Y-STR multiplex for familial searching.

The DNATyper™Y26 PCR Amplification kit, which including 26 low-medium mutating Y-STRs, is designed for Y-STR familial searching casework. The kit combines nine new Y-STR loci in addition to the 17 Y-STR loci from the commercially available AmpFlSTR®Yfiler® kit. The validation of the DNATyper™Y26 kit was performed in terms of technical index, including accuracy, stability, species specificity, sensitivity, adaptability for various samples, and mixture. Further, mutations of the 26 Y-STRs were analyzed by 1167 DNA-confirmed father-son pairs, and the results indicated that these loci had low or medium mutation rates. Furthermore, these Y-STRs loci were also tested in 1072 random male samples from Henan, Shanxi, Inner Mongolia, and Chongqing in China, showing their high power for forensic discrimination in the Chinese population. Thus, the DNATyper™Y26 PCR Amplification kit is a powerful tool for 'Y-STRs familial searching' in actual sexual-assault cases, indicating its unique advantage in familial searching due to Y-STR loci with only low-medium mutation rates.

Massively Parallel Sequencing of Forensic STRs Using the Ion Chef™ and the Ion S5™ XL Systems.

Next-generation sequencing (NGS) has been used to genotype forensic short tandem repeat (STR) markers for individual identification and kinship analysis. STR data from several NGS platforms have been published, but forensic application trials using the Ion S5™ XL system have not been reported. In this work, we report sensitivity, reproducibility, mixture, simulated degradation, and casework sample data on the Ion Chef™ and S5™ XL systems using an early access 25-plex panel. Sensitivity experiments showed that over 97% of the alleles were detectable with down to 62 pg input of genomic DNA. In mixture studies, alleles from minor contributors were correctly assigned at 1:9 and 9:1 ratios. NGS successfully gave 12 full genotype results from 13 challenging casework samples, compared with five full results using the CE platform. In conclusion, the Ion Chef™ and the Ion S5™ XL systems provided an alternative and promising approach for forensic STR genotyping.

Isolating Sperm from Cell Mixtures Using Magnetic Beads Coupled with an Anti-PH-20 Antibody for Forensic DNA Analysis.

Vaginal swabs taken in rape cases usually contain epithelial cells from the victim and sperm from the assailant and forensic DNA analysis requires separation of sperm from these cell mixtures. PH-20, which is a glycosylphosphatidylinositol-anchored hyaluronidase located on the head of sperm, has important functions in fertilization. Here we describe a newly developed method for sperm isolation using anti-PH-20 antibody-coupled immunomagnetic beads (anti-PH-20 IMBs). Optical microscopy and scanning electron microscopy showed the IMBs recognized the head of sperm specifically and exhibited a great capacity to capture sperm cells. However, we found it necessary to incubate the IMB-sperm complex with DNase I before sperm lysis in order to remove any female DNA completely. We compared the sensitivity of anti-PH-20 IMBs in sperm and epithelial cell discrimination to those coated with a different anti-sperm antibody (anti-SP-10, anti-ADAM2 or anti-JLP). Only the anti-PH-20 IMBs succeeded in isolating sperm from cell mixtures at a sperm/epithelial cell ratio of 103:105. Further, our method exhibited greater power and better stability for sperm isolation compared to the traditional differential lysis strategy. Taken together, the anti-PH-20 IMB method described here could be effective for the isolation of sperm needed to obtain a single-sourced DNA profile as an aid to identifying the perpetrator in sexual assault cases.

A new strategy for sperm isolation and STR typing from multi-donor sperm mixtures.

Mixed semen stains from multiple contributors are challenging samples in sexual assault casework, and it is crucial to obtain the DNA profiles of different donors to allow the evidence to play an important role in investigations and judicial proceedings. Current standard procedures, including preferential lysis, are incapable of separating single-source sperm from multiple male donors. Mixed profiles are often obtained and may not directly exclude or identify suspects. In this case, computational methods for mixture interpretation are often used, which rely on different types of calculation models. Here, we explored a new strategy for sperm cell isolation and detection from mixtures. It is a direct way to obtain genotypes of different sperm donors compared to computation-based mixture interpretation. Laser capture microdissection (LCM) and low volume-PCR (LV-PCR) were used for single sperm isolation and detection. The platform was sensitive; profiling of a single sperm cell generated a minimum of 13-16 loci in 73.1% of Y short tandem repeat (Y-STR) assays. A new Y-STR and autosomal STR multiplex system (YA-STR) were optimized by the combination of the Y-STR locus and 10 autosomal STR (auto STR) loci. The Y-STR locus acted as a tag to discriminate profile groups from different donors. Subsequently, consensus auto STR profiles of various persons could be received. The accuracy and availability of this method were evaluated on a three-donor semen mixture and found to be effective for the resolution of a multi-donor sperm mixture.

Construction of a library of cloned short tandem repeat (STR) alleles as universal templates for allelic ladder preparation.

Short tandem repeat (STR) genotyping methods are widely used for human identity testing applications, including forensic DNA analysis. Samples of DNA containing the length-variant STR alleles are typically separated and genotyped by comparison to an allelic ladder. Here, we describe a newly devised library of cloned STR alleles. The library covers alleles X and Y for the sex-determining locus Amelogenin and 259 other alleles for 22 autosomal STR loci (TPOX, D3S1358, FGA, D5S818, CSF1PO, D7S820, D8S1179, TH01, vWA, D13S317, D16S539, D18S51, D21S11, D2S1338, D6S1043, D12S391, Penta E, D19S433, D11S4463, D17S974, D3S4529 and D12ATA63). New primers were designed for all these loci to construct recombinant plasmids so that the library retains core repeat elements of STR as well as 5'- and 3'-flanking sequences of ∼500 base pairs. Since amplicons of commercial STR genotyping kits and systems developed in laboratories are usually distributed from 50 to <500 base pairs, this library could provide universal templates for allelic ladder preparation. We prepared three different sets of allelic ladders for this locus TH01 and an updated version of an allelic ladder for the DNATyper(®)19 multiplex system using these plasmids to confirm the suitability of the library as a good source for allelic ladder preparation. Importantly, the authenticity of each construct was confirmed by bidirectional nucleotide sequencing and we report the repeat structures of the 259 STR alleles. The sequencing results showed all repeat structures we obtained for TPOX, CSF1PO, D7S820, TH01, D16S539, D18S51 and Penta E were the same as reported. However, we identified 102 unreported repeat structures from the other 15 STR loci, supplementing our current knowledge of repeat structures and leading to further understanding of these widely used loci.

[Preliminary application of PCR-based assay for the detection of Neospora caninum in bovine aborted fetus].

OBJECTIVE: To establish a PCR diagnostic method based on Nc-5 gene of Neospora caninum, for being used to detect Neospora in brain tissues of bovine aborted fetus. METHODS: Specific primers were designed and synthesized based on the reported Nc-5 gene of N. caninum (GenBank Accession No. AY459289). Using genomic DNA from N.caninum as templates, Nc-5 gene was amplified by PCR. The PCR product was cloned into pMD18-T vector, transformed into Escherichia coli JM109 and then sequenced. To evaluate the specificity of the PCR, genomic DNA of Theileria annulata, Babesia bovis, Toxoplasma gondii, Leishmania donovani and standard strain of N. caninum were used as a template in the PCR. For determining the detection limit of amplification procedure, PCR was run on a dilution series of genomic DNA from N. caninum (1.562 5-200 ng/ml). Brain tissue samples of 32 aborted fetuses were detected by PCR-based assay, and 23 blood samples from mothers were tested by ELISA. RESULTS: The amplified DNA fragment (350 bp) had a high identity of 98% with the Nc-5 gene sequence of N. caninum (GenBank Accession No. AY459289). The PCR was specific for N. caninum and allowed the detection of 3.125 pg DNA of the parasite, while no amplification occurred with the other four species of protozoa. PCR-based assay and ELISA showed a positive rate of 18.8% (6/32) and 17.4% (4/23) of the samples tested, respectively. Moreover, all the 4 antibody positive samples showed PCR positive. There is no significant difference between the two assays (P > 0.05). CONCLUSION: PCR diagnostic method is promising in detecting Neospora infection in brain tissues of aborted bovine.

[Study of DNA identification in burned bones].

OBJECTIVE: For the purpose of solving a problems of DNA testing of burned bones. METHODS: We present a novel strategy to obtain DNA from burned bones based on the use of cetyltrimethylammonium bromide (CTAB) lysis buffer and isoamyl alcohol-chlorophorm extraction with subsequent DNA purification using the DNA IQ System. RESULTS: The methods were found to be effective in removing the PCR inhibitors from the burned bone. Then the extracted DNA was successfully genotyped by using the florescence labeling STR multiplex method. CONCLUSION: The results of this research will assist forensic scientists in the identification of DNA from victims whose bodies underwent significant trauma or burning, precluding the utilization of traditional forensic DNA identification techniques.