Microchip-based forensic short tandem repeat genotyping.

Cyanoaclyrate (CA) fuming has commonly been used to detect latent fingerprints and was reported in most cases not to adversely affect short tandem repeat (STR) typing. However, the effect of the CA fuming on DNA extraction efficiency was not reported. In this study, the DNA quantity of CA-fumed samples was measured by real-time PCR assay and STR typing was performed. Twenty microliters of saliva and 3 μl of blood were smeared on glass slides and polyethylene terephthalate (PET) sheets. The samples were treated with (1) CA, (2) CA and ultraviolet light (UV, 254 nm) irradiation, and (3) CA, 7-benzylamino-4-nitrobenz-2-oxa-1,3-diazole (BBD) and Polilight (450 nm) irradiation. The amounts of DNA extracted from the samples were quantified using real-time PCR assay and STR typing was performed. The saliva samples on the glass slides were covered with a large amount of CA, and the average of their DNA concentrations was reduced to 0.43 ng/μl, while that of non-treatment control was 0.75 ng/μl. Similar results were obtained from the saliva samples on the PET sheets. A Double swab technique did not improve the recovery of the DNA quantities. On the other hand, blood samples that were hardly covered with the CA had almost the same DNA quantities as those of the non-treatment control. Concerning the post treatment methods, the UV irradiation drastically reduced the DNA quantities, but the BBD and the Polilight hardly reduced them. Full STR profiles were obtained from all of the saliva and blood samples except for the samples irradiated with the UV. Ten PET bottle samples were fumed with the CA gas as the simulated casework samples, and full STR profiles were obtained from 8 out of 10 samples. In conclusion, DNA quantity of saliva samples was reduced by CA fuming probably due to the covering of a large amount of the CA; however, the CA fuming did not affect STR typing adversely.

Identification of a rare tri-allelic inheritance at the D8S1179 locus in a case of paternity testing

Postmortem perfusion is a procedure which provides in-vivo fixation of the human body and prevents organ and tissue decomposition after biological death occurs. Formaldehyde-based embalming solutions influence nucleic acid degradation, which reduces the quality and quantity of DNA extracted and the effectiveness of short tandem repeat (STR) typing. This research is a 1-year study aimed at determining the timeframe within which viable DNA profiles can be generated from different tissue types, post-embalming. Samples from the bone marrow (tibia), trapezius and quadriceps muscles, liver, and brain tissues were collected before embalming and at seven time points post-embalming. DNA extraction and quantification were performed on each sample to assess whether sufficient DNA was available for testing. Samples with DNA quantities over 0.05 ng/μL underwent amplification and STR analysis to produce DNA profiles. Quantification values and profile quality were compared across tissue types and time points. Results showed that prolonged exposure to embalming solutions significantly reduced DNA quality, leading to less discernible profiles. Statistically significant differences were detected among all tissue types at all time periods. Liver and brain samples retained more DNA than bone marrow, trapezius, and quadricep muscle samples. Qualitative evaluation of ski slope patterns in liver STR profiles demonstrated increasing signal degradation over time and was consistent across loci, suggesting predictable fragment length-dependent decline. This study suggests that after embalming, the liver is the preferential tissue for recovering DNA for forensic analysis, as the liver samples were the most robust and the only sample type to yield DNA profiles up to 1 year.

Multiplex STR typing of aortic tissues from unidentified cadavers

Identification of the perpetrator among identical twins using next-generation sequencing technology: A case report

Massively parallel sequencing of 10 autosomal STRs in Chinese using the ion torrent personal genome machine (PGM)

Aim. We tested the performance of two real-time polymerase chain reaction (PCR) human quantification kits (Human Quantifiler (Applied Biosystems) and Investigator Quantiplex kit (Qiagen)) and forensic identification short tandem repeat (STR) Investigator ESSplex Plus (Qiagen) kit on approximately 70 years old skeletal remains. Methods. We analysed 54 bones and teeth excavated from Second World War mass graves in Slovenia. Genomic DNA was obtained from 0.5 g of bone or tooth powder after total demineralization. The DNA was purified in a Biorobot EZ1 (Qiagen) device. The same extract was used for quantification and STR typing with all three kits using the amplification conditions recommended by the manufacturers. Results. In almost two thirds of the samples the results of quantification were up to 6 times higher using the Human Quantifiler than using the Investigator Quantiplex kit, because of the degradation of DNA in old skeletal remains and amplification of shorter DNA fragment with the Human Quantifiler kit. The autosomal STR typing with the Investigator ESSplex Plus kit was successful in 52 out of the 54 samples, which represent a 96% success rate. Conclusion. The commercially available Investigator ESSplex Plus kit can be used for STR typing of old skeletal remains with the DNA extraction method optimised in our laboratory and without any changes to the manufacturers' PCR amplification protocols. The Human Quantifiler kit and Investigator Quantiplex kit together can be used for estimation of the degree of DNA degradation in compromised old bone samples.

Short tandem repeats (STRs) are the most popular markers for human identification in forensics. These markers can be easily analyzed through a multiplex polymerase chain reaction and electrophoresis and provide high discrimination power. However, in STR analysis, several atypical phenomena can be observed such as allelic dropouts, drop-ins, or imbalance, which may be due to DNA polymerase slippage or DNA degradation effects. The observed atypical STR profiles can also provide information for mixed DNA samples or chromosomal abnormalities. In this study, we report a case of mosaicism detected in routine casework of paternity testing. Hair samples from a phenotypically normal male were tested, and the result presented a typical STR profile of a female for the amelogenin gene (XX). Through chromosome analysis using peripheral blood, it was found that 45,X/46,XY mosaicism resulted in the discrepancy between the genotype and the phenotype. In addition, the amount of Y chromosome detected was particularly low in hair compared to that in blood. This study shows that mosaicism can make interpretation difficult during STR analysis and suggests that sample types and repeated analysis should be considered even for routine STR testing.

Although progress has been seen for low template DNA (LT-DNA) analysis in the last few years, the obtainment of full short tandem repeat (STR) profiles remains challenging in forensic genetics. Preamplification treatment could help increase the recovery of genetic information in STR typing. In this study, an efficient amplification method was investigated, which shows that the application of the abasic site in a single primer for targets could efficiently delete the primer-binding site of PCR products to achieve semi-linear amplification (abSLA PCR). Further, the position of abasic sites located at 8th to 10th from the 3’ end of the primers could facilitate PCR amplification. A significant increase in the recovery of STR loci could be obtained using a 4-plex STR pre-amplification (D8S1179, D21S11, D7S820, and CSF1PO) coupled to Identifiler Plus kit in low template of genomic DNA or single cells. Altogether, the application of abSLA amplification method can facilitate conventional STR typing in LT-DNA analysis.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-19847-1.

Background: In forensic field, for establishing the paternity of disputed offspring, STR typing (autosomal, and Y typing) are commonly used to dissolve the cases. Short tandem repeat (STR) technology is used to evaluate specific regions (loci) within nuclear DNA. Variability in STR regions can be used to distinguish one DNA profile from another. The Federal Bureau of Investigation (FBI) uses a standard set of 13 specific STR regions for CODIS. Aim of the study: The present work was done to find out the biological father of child in a case where mother was killed by her husband for the doubt of her pregnancy. Methods: After taking blood samples from the murdered mother, the baby and the killer husband, Autosomal STRs Using AmpFlSTR Identifiler Plus PCR Amplification Kit and Y-STR analysis using AmpFlSTR Yfiler PCR Amplification Kit were done to solve the disputed paternity case. Results: After the analysis of DNA STRs profiles of autosomal and Y-chromosome markers, the dead fetus was proved to be the son of the killer father. Conclusions: Autosomal STR multiplex analysis combined with Y-STR analysis help to reach a conclusion in disputed paternity cases with greater confidence. Keywords: Disputed; Paternity; Multiplex; Short tandem reprats; Y-STR; Analysis

Short tandem repeat (STR) typing has become the standard technique in forensic methodology for the identification of unknown samples. National DNA databases have been established that contain STR genotypes for intelligence purposes. Due to their success, national DNA databases have been growing so fast that the number of advantageous matches may become a logistic problem for the analysts. This is especially true for partial STR profiles as they display reduced discrimination power. To overcome this drawback, modified versions (so-called mini-STRs) of existing loci were introduced as well as new loci to improve the information content of (partial) STR profiles. We pursue an alternative approach that makes use of nucleotide variation within the amplified STR fragments, which can be discerned by mass spectrometry. We have developed an assay that determines molecular masses from crude STR amplicons which were purified and separated by a liquid chromatographic system directly hyphenated to an electrospray ionization mass spectrometer. We present here new population data of forensically relevant STRs in Khoisan and Yakut populations. These autochthonous groups were selected as they may harbor additional STR alleles that are rare or unobserved in modern humans from cosmopolitan areas, especially for the Khoisan, which are known to represent a very ancient human population. The analysis of the molecular mass of STRs offered a widened spectrum of allele variability escorted by enhanced forensic use. Thus, established STR data derived from fragment size analysis can still be used in casework or in the context of intelligence databasing.

Assessing PreCR™ repair enzymes for restoration of STR profiles from artificially degraded DNA for human identification.

The analysis and profiling of short tandem repeat (STR) loci is routinely used in forensic genetics. Current methods to investigate STR loci, including PCR-based standard fragment analyses and capillary electrophoresis, only provide amplicon lengths that are used to estimate the number of STR repeat units. These methods do not allow for the full resolution of STR base composition that sequencing approaches could provide. Here we present an STR profiling method based on the use of the Roche Genome Sequencer (GS) FLX to simultaneously sequence multiple core STR loci. Using this method in combination with a bioinformatic tool designed specifically to analyze sequence lengths and frequencies, we found that GS FLX STR sequence data are comparable to conventional capillary electrophoresis-based STR typing. Furthermore, we found DNA base substitutions and repeat sequence variations that would not have been identified using conventional STR typing.

Establishing an integrated pipeline for automatic and efficient detection of trace DNA encountered in forensic applications

Single sperm cell isolation by laser microdissection