Advances in forensic DNA quantification: a review.

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.

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.

Isolation of DNA from skeletonised human remains can be problematic. In addition to DNA degradation, enhanced by high temperature and humidity, there are often potent polymerase chain reaction (PCR) inhibitors present within the samples. It is therefore important to extract the maximum amount of available DNA whilst removing any amplification inhibitors that may be present.Whilst real-time PCR methods are available for quantification and detection of PCR inhibitors the information received is limited as real-time PCR targets amplicons that are much smaller than those typically targeted in forensic analysis. To gain more information on the quality of extracted DNA a new multiplex PCR assay comprising a 4-plex targeting amplicons of 70 base pairs (bp), 194 bp, 305 bp and 384 bp along with two Internal Amplification Contols (IACs) of 90 bp and 410 bp was developed. This multiplex was optimised so that it worked with template amounts ranging between 0.10 ng and 200 ng; partial profiles were obtained with as little as 0.02 ng. The IACs were effective in detecting PCR inhibitors.The multiplex also assessed as a quantification tool. Plotting peak height compared to input DNA of a standard dilution series produced a coefficient of determination (R2) of 0.8308. The multiplex was found to provided reasonable estimates of DNA concentration, when the sample concentration was between 12.5 – 100 ng; relative standard deviations were all below 10% in this range for 30% of tested samples. However, real-time PCR proved to be more precise and was used in the rest of the study for the purposes of quantification.In forensic cases bones and teeth often provide some of the most challenging samples to extract good quality DNA. Using the optimised multiplex to assess the quality of DNA extracts five extraction methods: ChargeSwitch® gDNA Plant Kit, DNA IQTM System Kit, DNeasy® Blood & Tissue Kit, PrepFiler® BTA Forensic DNA Extraction Kit and phenol-chloroform-isoamyl alcohol extaction methods were assessed for their capability for extracting clean DNA from bone samples. Prior to the main experimentation several evaluation studies were carried out to optimise the methods being used. Based on the results, decalcification was not used for any of the extractions as non-decalcified extracts contained higher amounts of DNA. For the phenol-chloroform-isoamyl alcohol extraction it was determined that whilst ethanol precipitation provided higher amounts of DNA, the extracts using Amicon 30kDa filters (Amicon ultra-0.5 centrifugal filter unit with ultracel-30 membrane) were cleaner. Based on poor results with degraded bone samples a pre-process technique was developed; these extractions started with 250 mg of pulverised bone sample which was then concentrated and cleaned up using Amicon 30kDa filters (Amicon ultra-2 ml centrifugal filters for DNA purification and concentration) before carrying out the standard extraction procedures.After optimisation of the extraction methods the comparison study showed that the phenol-chloroform-isoamyl alcohol extraction method produced the highest DNA yields with both fresh and degraded bone samples, followed by DNeasy® Blood & Tissue Kit, ChargeSwitch® gDNA Plant Kit, PrepFiler® BTA Forensic DNA Extraction Kit and DNA IQTM System Kit. However, all produced DNA that could be amplified and did not contain any inhibition.Another application of the multiplex was to assess the effectiveness of different DNA preservation methods by examining the amount and quality of DNA recovered after preservation. Five methods: cell lysis solution (with 1% sodium azide), dehydration / freeze drying, ethanol (96%), freezing and room temperature storage were used to study the effectiveness of preservation methods on fresh and three-month old decomposed pig bone samples which were preserved for 6 weeks, 6 months and 1 year. The results showed that freezing is the best preservation method for both fresh and degraded bone samples for long-term storage followed by ethanol (96%), dehydration / freeze drying and room temperature storage. However, full profiles were obtained from both fresh and degraded bone samples from all methods, except cell lysis solution (with 1% sodium azide). Cell lysis solution (with 1% sodium azide) preservation method tended to be good for short-term storage but with the long-term preservation, less DNA yield was obtained and also the electropherograms showed higher levels of DNA degradation.Finally, using the optimised DNA extraction methods, the multiplex was tested using forensic samples comprising of 30 bone samples from casework in Malaysia and simulated body fluid evidences subjected to environmental insult in the United Arab Emirates. The application illustrated the effectiveness of the multiplex to identify PCR inhibitors and identify DNA degradation, providing supplementary information to real-time PCR.

Micro total analysis system (μTAS) or lab-on-a-chip (LOC) technology has advanced over decades, and the high performance for chemical and biological analysis has been well demonstrated with advantages of low sample consumption, rapid analysis time, high-throughput screening, and portability. In particular, μTAS or LOC based genetic applications have been extensively explored, and the short tandem repeat (STR) typing on a chip has garnered attention in the forensic community due to its special use for human identification in the field of mass disaster and missing person investigation, paternity testing, and perpetrator identification. The STR typing process consists of sample collection, DNA extraction, DNA quantitation, STR loci amplification, capillary electrophoretic separation, and STR profiling. Recent progress of microtechnology shows its ability to substitute the conventional analytical tools, and furthermore demonstrates total integration of the whole STR processes on a single wafer for on-site STR typing. In this review article, we highlighted some representative results for fluorescence labeling techniques, microchip-based DNA purification, on-chip polymerase chain reaction (PCR), a capillary electrophoretic microdevice, and a fully integrated microdevice for STR typing.

Developmental validation of the Quantifiler® HP and Trio Kits for human DNA quantification in forensic samples

Multiplex STR typing of aortic tissues from unidentified cadavers

Monitoring of Hematopoietic Chimerism by Real-Time Quantitative PCR of Single Nucleotide Polymorphisms In Samples with Low DNA Quantities.

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

Genotyping of biological samples is now routinely performed in human identication (HID) laboratories for applications like paternity testing, forensic casework, DNA databasing, missing persons testing, lineage studies, identication of remains, and so forth. e genotyping protocol comprises sample collection, extraction of DNA from the biological sample, quantication of the DNA, amplication of target loci, identication of amplied products, and the analysis of results. Extraction of DNA is the rst and most critical step of sample processing simply because a better quality and higher quantity of DNA leads to a higher quality, and likely more complete, genotype. Isolation of DNA from forensic samples is a challenging process that creates bottlenecks in the sample-processing work-ow. e challenges are primarily due to the nature of biological samples. Unlike most clinical samples, the biological samples processed in forensic laboratories vary widely and include biological uids (saliva, blood, and semen), tissues, hair, bone, tooth, nail, and so forth. Moreover, body uids can be deposited on a wide range of substrates (rather unlimited), mixed with inhibitors of the polymerase chain reaction (PCR), exposed to varying environmental conditions, or subjected to uncontrolled degradation, and they are oen present in limited quantities. Even the single-source samples processed for DNA databasing exhibit variation in the type of body uid (typically blood or saliva) and substrates. e spectrum of substrates on which biological samples get deposited expands dramatically for forensic evidence samples. Examples of the most common PCR inhibitors found in forensic samples include hematin from blood, humic acids from soil, textile dyes such as indigo from denim, calcium from bones, melanin from soil, and tannins from leather. e amount of DNA isolated from forensic samples cannot be predicted ahead of extraction because of the nature of these samples.

Simple SummaryThe cloacal anatomy is unique because the fecal, urinary, and reproductive tracts converge into one orifice. Therefore, sampling for microbiome research can be difficult in birds, especially in agricultural production settings where it may not be feasible to sample the intestines, and cloacal swabs are often used. There is a need to evaluate laboratory methods for 16S rRNA sequencing in cloacal swab samples to ensure reproducible and trustworthy downstream results. We compared four DNA extraction methods from two commercially available magnetic-based DNA extraction kits. Mock communities and negative controls were included for each method and subjected to 16S rRNA sequencing. While extraction quality and yield differed between each extraction method, overall sequencing results were not affected, including alpha and beta diversity. Positive and negative controls are an important aspect of microbiome science and our findings lend guidance to future microbiome research in poultry.As the applications of microbiome science in agriculture expand, laboratory methods should be constantly evaluated to ensure optimization and reliability of downstream results. Most animal microbiome research uses fecal samples or rectal swabs for profiling the gut bacterial community; however, in birds, this is difficult given the unique anatomy of the cloaca where the fecal, urinary, and reproductive tracts converge into one orifice. Therefore, avian gut microbiomes are usually sampled from cloacal swabs, creating a need to evaluate sample preparation methods to optimize 16S sequencing. We compared four different DNA extraction methods from two commercially available kits on cloacal swabs from 10 adult commercial laying hens and included mock communities and negative controls, which were then subjected to 16S rRNA amplicon sequencing. Extracted DNA yield and quality, diversity analyses, and contaminants were assessed. Differences in DNA quality and quantity were observed, and all methods needed further purification for optimal sequencing, suggesting contaminants due to cloacal contents, method reagents, and/or environmental factors. However, no differences were observed in alpha or beta diversity between methods. Importantly, multiple bacterial contaminants were detected in each mock community and negative control, indicating the prevalence of laboratory and handling contamination as well as method-specific reagent contamination. We found that although the extraction methods resulted in different extraction quality and yield, overall sequencing results were not affected, and we did not identify any method that would be an inappropriate choice in extracting DNA from cloacal swabs for 16S rRNA sequencing. Overall, our results highlight the need for careful consideration of positive and negative controls in addition to DNA isolation method and lend guidance to future microbiome research in poultry.

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

The identification of putrefied bodies is a common task in forensic routine work. The deceased are usually identified by dental records, fingerprinting, or--in cases where no such data are available--DNA analysis. However, with progressive putrefaction, DNA integrity is rapidly decreasing. Genetic analysis may then be greatly impaired, if not impossible. The aim of our study was to establish an efficient procedure to successfully extract and amplify DNA from soft tissues of bodies in different stages of putrefaction. Soft tissues-unlike teeth or bones-usually allow the application of fast and easy-to-use extraction protocols. DNA was extracted from different tissues (aorta, kidney, liver, and skeletal muscle) taken at autopsy using a commercially available DNA extraction kit, and DNA quality and quantity were controlled by agarose gel electrophoresis and real-time polymerase chain reaction (PCR). Presence of mitochondrial DNA was tested using a highly sensitive duplex PCR. Short tandem repeat analysis was done using the AmpFlSTR Identifiler kit. Additionally, mitochondrial DNA sequencing was performed. After DNA extraction from at least two different tissues-preferably the kidney and the aorta-with the extraction kit based on the Nucleobond method, a successful amplification of at least eight loci was possible in 17 out of 18 cases, and 12 or more loci could be amplified in 15 cases.

A simple alkali lysis method for Plasmodium falciparum DNA extraction from filter paper blood samples

Targeted capture massively parallel sequencing is increasingly being used in clinical settings, and as costs continue to decline, use of this technology may become routine in health care. However, a limited amount of tissue has often been a challenge in meeting quality requirements. To offer a practical guideline for the minimum amount of input DNA for targeted sequencing, we optimized and evaluated the performance of targeted sequencing depending on the input DNA amount. First, using various amounts of input DNA, we compared commercially available library construction kits and selected Agilent’s SureSelect-XT and KAPA Biosystems’ Hyper Prep kits as the kits most compatible with targeted deep sequencing using Agilent’s SureSelect custom capture. Then, we optimized the adapter ligation conditions of the Hyper Prep kit to improve library construction efficiency and adapted multiplexed hybrid selection to reduce the cost of sequencing. In this study, we systematically evaluated the performance of the optimized protocol depending on the amount of input DNA, ranging from 6.25 to 200 ng, suggesting the minimal input DNA amounts based on coverage depths required for specific applications.

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