A comparison of five DNA extraction methods from degraded human skeletal remains

Extracting DNA from degraded human remains poses a challenge for any forensic genetics laboratory, as it requires efficient high-throughput methods. While little research has compared different techniques, silica in suspension has been identified in the literature as the best method for recovering small fragments, which are often present in these types of samples. In this study, we tested five DNA extraction protocols on 25 different degraded skeletal remains. Including the humerus, ulna, tibia, femur, and petrous bone. The five protocols were organic extraction by phenol/chloroform/isoamyl alcohol, silica in suspension, High Pure Nucleic Acid Large Volume silica columns (Roche), InnoXtract™ Bone (InnoGenomics), and PrepFiler™ BTA with AutoMate™ Express robot (ThermoFisher). We analysed five DNA quantification parameters (small human target quantity, large human target quantity, human male target quantity, degradation index, and internal PCR control threshold), and five DNA profile parameters (number of alleles with peak height higher than analytic and stochastic threshold, average relative fluorescence units (RFU), heterozygous balance, and number of reportable loci) were analysed. Our results suggest that organic extraction by phenol/chloroform/ isoamyl alcohol was the best performing method in terms of both quantification and DNA profile results. However, Roche silica columns were found to be the most efficient method

QUANTIFILER®TRIO DNA method performance in a collection of ancient samples

Trabalho científico apresentado sob a forma de Poster no evento 9th ISABS Conference on Forensic Anthropologic Genetics and Mayo Clinicm Bol, Island of Brac, Croacia, 2015During the past few years significant progress has been made in solving technical challenges associated with STR profiling including the ability to analyze degraded DNA and low amounts of DNA. The result of these changes is that useful STR profiles can now be obtained from previously untypeable forensic DNA samples. Analysis of DNA from ancient material represents an important role in molecular anthropology, although there are many limitations concerning low DNA quantity and/or degraded DNA, and/or PCR inhibitors. These factors can make it difficult to decide whether to continue with STR analysis, which STR panel to use and how much DNA to add to PCR reaction. With all these constraints, DNA quantification represents an important tool to decide which method will follow in order to improve workflow and have good results in less time-consuming. The Quantifiler® Trio DNA method provides a quality index (QI) to detect the presence of degraded DNA along with PCR inhibitors.This guide allows the selection of the optimal short tandem repeat (STR) analysis chemistry (autosomal, or miniSTR) and streamlines the workflow while increasing downstream analysis success rates. In order to compare DNA quality from different extraction methods, samples from 46 exhumed Middle Ages individuals were extracted with modified phenol-chloroform method and also PrepFiler Express BTA™ method. DNA was quantified with Quantifiler® Trio DNA Quantification in an Applied Biosystems® 7500 Real-Time PCR System. Results were analyzed and allow us to point Quantifiler® Trio method as an important tool in pre-STR typing methods in ancient samplesN/

Application of enhancement strategies for the improvement of discriminating forensic DNA profiles from human bones

Although DNA profiling techniques are considered a powerful method for identification, problems arise when low quantity and/or quality DNA is tested. Analyzing samples with low template DNA using standard genotyping techniques like STR typing can yield no or an incomplete profile, making conclusive identification nearly impossible. One challenging forensic sample material is bone. For example, DNA within bone can be degraded due to harsh environmental conditions even when the structure of the bone appears to be relatively well preserved. Consequently, the amount of usable DNA in bones can be limited which complicates downstream applications for DNA profiling. This study investigated techniques that would improve obtaining a discriminating DNA profile from human bones. First, the performances of three commercial DNA extraction kits were evaluated for the recovery of genomic DNA from human bones. The PrepFiler® BTA Forensic DNA Extraction Kit recovered the highest DNA yield according to qPCR data and was used for additional bone extractions. Next, purified DNA from a total of 12 bone samples was subject to genotyping methods using capillary electrophoresis (CE) as well as massively parallel sequencing (MPS) to determine which strategy would produce the most discriminating DNA profile. The genotyping techniques evaluated were: CE-based STR analysis with the GlobalFiler® PCR Amplification Kit, whole genome amplification (WGA) with the REPLI-g® Mini Kit for improved CE-based STR detection, and MPS for STR/SNP analysis using the Ion PGM™ and MiSeq® FGx™ platforms. Random match probabilities were calculated to determine the discriminatory power of the resulting DNA profiles. Although the Ion PGM™ SNP profiles had the highest RMPs, the GlobalFiler® STR profiles produced similar discriminatory power. Considering the time and labor required for MPS, one could argue STR analysis using traditional CE may be better suited for DNA profiling of challenging bone samples. However, the MPS technologies provide additional information that CE-methods can’t, such as Y-haplogroup and biogeographical ancestry predictions from SNP analyses

The Effect Of Sandy Surfaces on Touch DNA

Touch DNA profiling is an important tool to solve the mystery of many cases, especially when other biological evidences cannot be found in crime scene. However, there are many variables that influence Touch DNA profiling such as recovery techniques and extraction. In addition, effect of environmental factors on items found outdoor such as sand can impact on the process. Therefore the aim of this experiment was to test how sandy surfaces can affect the recovery of Touch DNA Profiling by validation two recovery methods and two extraction kits that are widely used in the DNA forensic field

Development and application of a PCR multiplex to assess the quality and quantity of forensic DNA extracts

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

Optimization of a method for the extraction of DNA from human skeletal remains for forensic casework

Obtaining full DNA profiles from bone can be challenging due to the inherently low quantity and quality of DNA. An efficient extraction protocol is required to maximize DNA recovery from bone samples while minimizing the coextraction of the PCR inhibitors naturally present in bone.DNA extraction from bone involves three discrete steps: demineralization, lysis, and purification. Demineralization involves incubating bone that has been pulverized into a powder in a chelating agent in order to sequester the divalent metal cations that comprise the mineral matrix of bone tissue. This is followed by a lysis step, in which the demineralized bone material is incubated in a buffer that disrupts the cellular membrane, releasing the DNA and intracellular components. Purification is performed after lysis to separate the DNA from proteins and cellular debris, as well as to remove PCR inhibitors.Several variables were examined in this study, including the use of three different lysis buffers, two different bone types, two different materials for the end caps and impactor bar used in the pulverization process, and the interactions between these variables. The effectiveness of each modification in the procedure was assessed using qPCR, which was used to measure mtDNA recovery in copies of mtDNA per gram of bone material, as well as by the assessment of inhibition using an internal positive control. The actual effectiveness of each lysis buffer varied considerably with bone types examined. In addition, a novel modification to the pulverization step was found to significantly enhance mtDNA recovery by reducing inhibition detected in samples.The findings of this work suggest that the current methodology is not fully optimized, and additional emphasis needs to be placed upon removing inhibitors but also investigating the effects of the bone lysate on the pH and ionic strength on the recovery of DNA from bone material. Preliminary work is presented from this study in developing a method for DNA extraction from human bone and elucidating the sources of inhibition that are introduced via the extraction method itself as well as those naturally present in bone tissue

DNA recovery from biological material on mini tapes using a simple extraction buffer and solid phase reversible immobilisation (SPRI) purification

In this study, we compare the performance of a simple PVP extraction method with a commercially available and widely used kit for recovering DNA from adhesive tapes. This novel method shows almost 60% higher DNA recovery from blood deposits on SceneSafe Fast™ minitapes when compared to the PrepFiler™ BTA Forensic DNA Extraction Kit. We also demonstrate how a simple modification of the magnetic bead-based purification step can lead to better recovery and removal of PCR inhibitors.<br/

Efficiency evaluation of the classic organic and new commercial approach in DNA extraction from bone samples using Qubit fluorometer and qPCR

UVOD: DNA u kosti nalazimo unutar stanica osteoblasta, osteocita i osteoklasta. Tijekom resorpcije kosti i stanične apoptoze, DNA dospijeva u izvanstanični matriks i veže molekule kolagena i hidroksiapatita. Za uspješnu izolaciju DNA iz kosti potrebno je primijeniti metode i reagense koji će osigurati specifično izdvajanje malih količina DNA materijala iz smjese drugih bioloških molekula, ostataka soli i organskih otapala. U tu svrhu ispitivana je učinkovitost klasične organske metode te QIAamp DNA Blood Mini i PrepFiler BTA Forensic Extraction komercijalnih kompleta. CILJ: Odrediti djelotvornost ispitivanih metoda za izolaciju DNA iz koštanog praha mjerenjem količine i kvalitete DNA na Qubit fluorometru i qPCR uređaju. MATERIJAL I METODE: Za istraživanje su upotrijebljeni poolirani uzorci koštanog praha. Izolacija DNA provedena je primjenom klasične organske fenol/kloroform metode te QIAamp DNA Blood Mini i PrepFiler Forensic DNA Extraction komercijalnih kompleta. Koncentracija DNA izmjerena je na Qubit fluorometru i qPCR uređaju pomoću Qubit High Sensitivity DNA i QuantiFiler Human Quantification DNA setova. REZULTATI: Najveći prinos DNA primijećen je u uzorcima izoliranima PrepFiler BTA komercijalnim kompletom. Slijedi ga QIAamp DNA Blood Mini komplet s nešto manjim koncentracijama DNA, a najmanje koncentracije DNA dobivene su klasičnom organskom metodom. Graf Linova koeficijenta konkordancije pokazuje jako mali stupanj korelacije i podudarnosti između mjerenja na Qubit fluorometru i Quant Studio 5 qPCR uređaju. ZAKLJUČAK: U usporedbi s klasičnom organskom metodom, silika kolone i selektivne magnetske čestice osiguravaju veću količinu i kvalitetu DNA materijala iz uzoraka kosti.INTRODUCTION: DNA in the bone is found in osteoblasts, osteocytes and osteoclasts. During bone resorption and cell apoptosis, DNA ends up in the extracellular matrix and binds to the molecules of collagen and hidroxyapatite. For a successful isolation of DNA from the bones, highly efficient methods and reagents should be employed to ensure the specific extraction of small amounts of DNA material, free from other biological molecules, residues of salts and organic solvents. For this purpose, we investigated the efficiency of the classic organic method, QIAamp DNA Blood Mini kit and PrepFiler BTA Forensic Extraction kit. GOAL: To determine the efficiency of the investigated methods for DNA isolation from bone powder, by measuring the amount and quality of DNA on the Qubit fluorometer and qPCR. MATERIALS AND METHODS: Pooled samples of bone powder were used for this study. DNA isolation was done using classical organic phenol / chloroform method, QIAamp DNA Blood Mini and PrepFiler Forensic DNA Extraction commercial sets. The concentration of DNA was measured on Qubit fluorometer and qPCR using Qubit High Sensitivity DNA and QuantiFiler Human Quantification DNA Kit. RESULTS: The highest DNA yield was found in samples isolated with PrepFiler BTA commercial kit. The QIAamp DNA Blood Mini Kit follows with slightly lower DNA concentrations, and the lowest DNA concentrations were obtained by classical organic method. Graph of Lin's concordance coefficient shows a very small correlation and correlation between Qubit fluorometer and Quant Studio 5 qPCR. CONCLUSION: Compared to the classical organic method, silica columns and selective magnetic particles provide greater quantity and quality of DNA material from bone samples

Extraction efficiency testing of degraded bone samples: Comparing four DNA extraction methods for downstream massively parallel sequencing applications

In recent years, investigative genetic genealogy (IGG), which involves the use of genealogical methods combined with DNA analysis to make potential familial matches, has become an important tool in solving cold and active cases. These cases can involve the identification of a perpetrator or the identification of missing persons. Estimates show that approximately 4,400 unidentified bodies are recovered each year, and up to one quarter of those individuals remain unidentified after one year. Traditionally, forensic DNA amplification methods have relied on the need to amplify 100-450 base-pair targets, specifically, short tandem repeats (STRs), for forensic profiles. With genetic genealogical approaches, smaller targets, such as single nucleotide polymorphisms (SNPs), have shown potential as tools for identification. Traditional extraction protocols for forensic DNA have focused on maximizing DNA recovery with the intent of amplifying larger STR targets. On the other hand, ancient DNA techniques have focused efforts on recovering smaller DNA fragments, like SNPs, and indeed have shown recovery of even highly degraded samples in excess of 400,000 years. This study aims to compare the extraction success and efficiency of one ancient DNA (aDNA) extraction technique from Rohland et al. (2018) and three forensic DNA extraction techniques, PrepFiler® BTA Forensic DNA Extraction Kit from Applied Biosystems, the Bone DNA Extraction Kit, Custom from Promega, and the InnoXtract™ from InnoGenomics, on compromised bone samples for the purposes of massively parallel sequencing (MPS). Quantitative PCR was used to compare the extraction performance of the protocols, while an MPS-based assay, the Ion AmpliSeq™ PhenoTrivium Panel, was used to assess informative characteristics, such as phenotype and biogeographic ancestry, for an investigation. The Rohland and modified PrepFiler protocols showed the most success in terms of DNA recovery and sequencing. These results show the utility of an ancient DNA extraction method in MPS research and the success of a widely used forensic method. The results of this study may add to the process of determining the most appropriate extraction method for massively parallel sequencing applications such as IGG in forensic contexts