DNA IQTM Research Articles

Improving DNA recovery and sample throughput using the PrepFiler™ Automated Forensic DNA Extraction Kit on two customised Tecan Fluent® 1080 Automated Workstations

One of the primary goals of forensic laboratories performing DNA recovery and profiling is obtaining a high-quality DNA extract from crime scene samples. This is largely dependent on the sensitivity and reliability of the DNA extraction chemistry utilised, as well as the liquid handling and contamination minimisation techniques employed. Automation of DNA extraction methods on large liquid handling platforms allows high-throughput laboratories to apply sensitive chemistries to reliably process a large number of samples, while minimising manual processes and cross-contamination.This study describes the first known implementation of the PrepFiler™ Automated Forensic DNA Extraction Kit on a Tecan Fluent® Gx 1080 Automation Workstation. Two Workstations were customised with the addition of novel “Safe Pipetting Modules” to eliminate sample crossover between wells, which is important in a forensic biology setting to reduce inadvertent DNA transfer. A comparison of DNA extraction efficiency between an optimised PrepFiler™ method and the DNA IQ™ System performed on a Perkin Elmer Janus® Integrator platform showed the optimised PrepFiler™ method consistently extracted a higher yield of DNA from a range of blood inputs, as well as blood and buccal swabs. The PrepFiler™ chemistry also more efficiently removed humic acid and haematin, reducing subsequent PCR inhibition. The subsequent implementation of the optimised PrepFiler™ method onto the Tecan Fluent® Gx workstations showed a further increase in sensitivity, with no evidence of DNA cross-contamination observed. However, the optimised PrepFiler™ method encountered difficulties extracting DNA from fabric substrates, with the PrepFiler Express™ chemistry extracting higher yields on the cartridge-based AutoMate Express™ System.Overall, this study demonstrated the Tecan Fluent® Gx 1080 Automation Workstation is a sensitive, reliable and robust method for DNA extraction using the PrepFiler™ Automated Forensic DNA Extraction Kit, and the addition of the novel Safe Pipetting Module makes this platform an attractive option for forensic biology laboratories where minimising inadvertent DNA transfer is of paramount importance.

Evaluation of a New DNA Extraction Method on Challenging Bone Samples Recovered from a WWII Mass Grave.

Bones and teeth represent a common finding in ancient DNA studies and in forensic casework, even after a long burial. Genetic typing is the gold standard for the personal identification of skeletal remains, but there are two main factors involved in the successful DNA typing of such samples: (1) the set-up of an efficient DNA extraction method; (2) the identification of the most suitable skeletal element for the downstream genetic analyses. In this paper, a protocol based on the processing of 0.5 g of bone powder decalcified using Na2EDTA proved to be suitable for a semi-automated DNA extraction workflow using the Maxwell® FSC DNA IQ™ Casework Kit (Promega, Madison, WI, USA). The performance of this method in terms of DNA recovery and quality was compared with a full demineralisation extraction protocol based on Qiagen technology and kits. No statistically significant differences were scored according to the DNA recovery and DNA degradation index (p-values ≥ 0.176; r ≥ 0.907). This new DNA extraction protocol was applied to 88 bone samples (41 femurs, 19 petrous bones, 12 metacarpals and 16 molars) allegedly belonging to 27 World War II Italian soldiers found in a mass grave on the isle of Cres (Croatia). The results of the qPCR performed by the Quantifiler Human DNA Quantification kit showed values above the lowest Limit of Quantification (lLOQ; 23 pg/µL) for all petrous bones, whereas other bone types showed, in most cases, lower amounts of DNA. Replicate STR-CE analyses showed successful typing (that is, >12 markers) in all tests on the petrous bones, followed by the metacarpals (83.3%), femurs (52.2%) and teeth (20.0%). Full profiles (22/22 autosomal markers) were achieved mainly in the petrous bones (84.2%), followed by the metacarpals (41.7%). Stochastic amplification artefacts such as drop-outs or drop-ins occurred with a frequency of 1.9% in the petrous bones, whereas they were higher when the DNA recovered from other bone elements was amplified (up to 13.9% in the femurs). Overall, the results of this study confirm that petrous bone outperforms other bone elements in terms of the quantity and quality of the recovered DNA; for this reason, if available, it should always be preferred for genetic testing. In addition, our results highlight the need for accurate planning of the DVI operation, which should be carried out by a multi-disciplinary team, and the tricky issue of identifying other suitable skeletal elements for genetic testing. Overall, the results presented in this paper support the need to adopt preanalytical strategies positively related to the successful genetic testing of aged skeletal remains in order to reduce costs and the time of analysis.

Comparison of three DNA extraction methods tested on illicit drug-related powders

The detection of human DNA on and within illicit drug preparations is novel and a focus of current research. Previous studies have indicated that certain drug-related powders present in illicit drug preparations can interfere with downstream DNA analysis when directly added to the PCR. Therefore, it is important to determine if these drug-related powders are effectively removed during the DNA extraction or whether traces of powder remain to interfere with DNA processing. Three extraction methods were selected to assess their efficiency at removing drug-related powders for downstream processes using DNA from both saliva and touch depositions. This is the first study to compare efficiencies of DNA extraction methods from drug-related powders. The extraction methods compared were the DNA IQ™ System, the QIAamp® DNA Investigator Kit and the combination of a simple lysis step followed by use of the Microcon® DNA Fast Flow device. Saliva was added to dimethylsulfone (DMS), nitrostyrene and PROSOLV® tablet mixture to determine the effect of powder type (based on solubility). Saliva was also added to 0, 50, 200 and 400 mg of DMS to determine the effect of an increase in DMS quantity. Trace DNA was deposited onto DMS using a worn glove approach. These samples were re-tested six months post-DNA deposition and profiled for further comparisons. Ten replicates were conducted for each condition with five replicates of saliva positive controls per method (n = 255 samples). A subset of samples was chemically analysed to determine if DMS was present in the final DNA eluant. The readily soluble DMS did not interfere with any of the extraction methods at lower amounts, however increasing the DMS to 400 mg reduced the relative DNA yields using the Microcon® and Investigator methods. The tablet mixture reduced the relative DNA yield of all three methods, however the nitrostyrene (which was relatively insoluble) only reduced the relative DNA yield of the DNA IQ™. The Investigator method performed the best with the trace samples, followed by the Microcon® method and then the DNA IQ™. DMS was detected in all extracts chemically analysed from the DNA IQ™ and Microcon®, whereas only one sample tested from the Investigator kit contained DMS in the extract and was in a relatively low amount compared to the other samples. Not one kit outperformed the others in all comparisons, however the Investigator kit was the most efficient overall at optimising the DNA yield whilst also removing the powders more effectively.

Differential Extraction with Purification via Organic/Microcon® and Promega DNA IQ™ Methods.

The differential extraction method allows for the separation of sperm cell DNA from non-sperm cell DNA by incorporating two separate lysis steps. This is crucial in forensic casework, as sexual assault samples frequently deal with a mixture of seminal fluid and other body fluids. After performing a differential lysis, DNA extraction can be completed through a variety of methods. In addition to the differential lysis, two methods will be described in this chapter for DNA purification: Organic (Phenol)/Microcon® purification and purification with the Promega DNA IQ™ System.

Optimization of DNA extraction and sampling methods for successful forensic microbiome analyses of the skin and saliva.

Microbiome studies have contributed to many fields, such as healthcare and medicine; however, these studies are relatively limited in forensics. Microbiome analyses can provide information, such as geolocation and ancestry information, when short tandem repeat (STR) profiling fails. In this study, methods for DNA extraction and sampling from the skin and saliva were optimized for the construction of a Korean Forensic Microbiome Database (KFMD). DNA yields were estimated using four DNA extraction kits, including two automated kits (Maxwell® FSC DNA IQ™ Casework Kit and PrepFiler™ Forensic DNA Extraction Kit, updated) and two manual kits (QIAamp DNA Mini Kit and QIAamp DNA Micro Kit) commonly used in forensic DNA profiling laboratories. Next-generation sequencing of the 16S rRNA V4 region was performed to analyze microbial communities in samples. The Bacterial Transport Swab with Liquid Media (NobleBio), two cotton swabs (PoongSung and Puritan), and nylon-flocked swabs (NobleBio and COPAN) were tested for DNA recovery. The PrepFiler and Maxwell kits showed the highest yields of 3.884ng/μL and 23.767ng/μL from the scalp and saliva, respectively. With respect to DNA recovery, nylon-flocked swabs performed better than cotton swabs. The relative abundances of taxa sorted by DNA extraction kits were similar contributions; however, with significant differences in community composition between scalp and saliva samples. Lawsonella and Veillonella were the most abundant genera in the two sample types. Thus, the Maxwell® FSC DNA IQ™ Casework Kit and nylon-flocked swab (NobleBio) were optimal for DNA extraction and collection in microbiome analyses.

Recovery of DNA from bone without demineralization

This study assessed the performance of five different DNA extraction methods for the recovery of DNA from bone: ChargeSwitch® gDNA Plant Kit, DNA IQ™ System Kit, DNeasy® Blood & Tissue Kit, PrepFiler® BTA Forensic DNA Extraction Kit and phenol-chloroform-isoamyl alcohol. DNA was extracted from pig rib and femur bones that was fresh, had undergone surface decomposition for three months, and had undergone surface decomposition for one year. Extracted DNA was analyzed using real-time PCR and amplification of an in-house PCR multiplex that assessed the quality and quantity of DNA and for the presence of inhibitors. The phenol-chloroform-based method consistently yielded the highest amounts of DNA and DNA IQ the lowest; however, all methods produced relatively high yields of DNA from both pig rib and femur samples that could be amplified without any detected inhibition. The data demonstrate that with reasonable quality bone samples any of the tested methods can isolate DNA that can be successfully analyzed. The effective use of internal PCR controls is also demonstrated.

Improved autosomal STR typing of degraded femur samples extracted using a custom demineralization buffer and DNA IQ™

Bone samples are utilized as a source of DNA in disaster victim identification and human remains investigations. However, DNA recovery from bones is time consuming and prone to contamination. A logical approach for postmortem identification is to validate efficient DNA extraction methods requiring less bone material using improved molecular kits with less hands-on time and workflows that facilitate faster turn-around time for processing. In this study, we evaluated DNA yield and amplification efficiency of DNA extracts using a new custom bone demineralization buffer (DMB; Promega) and extracted via manual and automated DNA IQ™ workflows. Including the demineralization step, the bone protocol can be completed in ∼4 h and even less with minimal sample handling when automated. Overall, a rapid and simple DNA extraction with improved allele recovery was validated using degraded bone samples exposed to tropical environments and post-disaster recovery as well as adverse conditions of embalming prior to internment.

Evaluation of the efficiency of Isohelix™ and Rayon swabs for recovery of DNA from metal surfaces.

We investigated the recovery and extraction efficiency of DNA from three metal surfaces (brass, copper, steel) relevant to forensic casework, and plastic (control) using two different swabbing systems; Rayon and Isohelix™ swabs, with sterile water and isopropyl alcohol respectively, as the wetting solutions. Twenty nanograms of human genomic DNA were applied directly to Isohelix™ and Rayon swabs; and to the metal and plastic substrates. All substrates were left to dry for 24h, followed by single wet swabbing and extraction with the DNA IQ™ System. DNA extracts were quantified using real time quantitative PCR assays with SYBR green chemistry. DNA was extracted from directly seeded Isohelix™ swabs with a high efficiency of 98%, indicating effective DNA-release from the swab into the extraction buffer. In contrast, only 58% of input DNA was recovered from seeded Rayon swabs, indicating higher DNA retention by these swabs. Isohelix™ swabs recovered 32 - 53% of DNA from metal surfaces, whilst the Rayon swabs recovered 11-29%. DNA recovery was lowest from copper and highest from brass. Interestingly, Rayon swabs appeared to collect more DNA from the plastic surface than Isohelix™ swabs, however, due to the lower release of DNA from Rayon swabs they returned less DNA overall following extraction than Isohelix™ swabs. These results demonstrate that DNA samples deposited on metal surfaces can be more efficiently recovered using Isohelix™ swabs wetted with isopropyl alcohol than Rayon swabs wetted with sterile water, although recovery is affected by the substrate type.

Casework direct kit as an alternative extraction method to enhance touch DNA samples analysis

Latent fingerprints are commonly found in crime scenes, and currently used in forensic analysis to obtain STR profiles from DNA recovered from finger contact. Analysis of STR profiles obtained from touch DNA has been very useful to elucidate crimes and the extraction method may be determinant for the recovery of genetic material collected from different surfaces. This study aimed to verify and compare the efficiency of two different extraction kits for processing touch DNA samples obtained from fingerprints deposited on computer keyboards, knife handles and exterior door handles and steering wheels of cars.One hundred and four experiments were conducted to simulate crime scenes and evaluate the efficiency of two extraction kits for touch DNA samples: the DNA IQ™ System and the Casework Direct Kit (both Promega Corporation). Each experiment was conducted with two individuals in order to obtain a mixture profile. The genetic material deposited was collected by double swab method (Sweet et al. 1997) and DNA quantification was conducted using Quantifiler Trio™ (ThermoFisher Scientific). Samples were amplified by PowerPlex® Fusion System kit (Promega).It was possible to obtain STR profiles for 32 (61.5%) out of the 52 extracted using DNA IQ and 51 (98.1%) out of the 52 extracted using the Casework Direct Kit. Samples extracted by DNA IQ had higher average of quantification values for long targets (>200bp) across all tested surfaces. That seems to be due to an incompatibility between the Quantifiler Trio and the Casework Direct Kit. Samples with positive quantification but without STR profile, as well as samples without quantification but with STR profiles were also observed.Statistical analysis showed that the Casework Direct Kit produced significantly more useful profiles than DNA IQ (p-value = 0.001), since these profiles had more STR markers with allelic correspondence to second donators present in the mixture.This study provides insights about the effect of different surfaces and extraction methods on recovery and generation of STR profiles. Limitations for the quantification step for these samples with a low quantity of DNA were highlighted as well. We concluded that the Casework Direct Kit was much more efficient for processing touch DNA samples than DNA IQ.

Evaluation of DNA Extraction Methods for Processing Fingerprint Powder-Coated Forensic Evidence.

In unison, fingerprinting and DNA analysis have played a pivotal role in forensic investigations. Fingerprint powders that are available on the market can come in a range of colors and with specific properties. This study evaluated the efficiency of DNA extraction from samples coated with 3 brands of fingerprint powders: Lightning, Sirchie, and SupraNano, covering a range of colors and properties. A total of 23 fingerprint powders were tested using the Chelex, Promega DNA IQ™, and Applied Biosystems™ PrepFiler™ DNA extraction protocols. The DNA IQ™ and PrepFiler™ methods extracted higher yields of DNA in comparison to Chelex, which also accounted for better quality of PowerPlex x00AE; 21 DNA profiles recovered. There were no signs of degradation or inhibition in the quantification data, indicating that samples returning low DNA yield was due to interference during DNA extraction and not PCR inhibition. DNA profiles were recovered from the majority of fingerprint powders with only a single powder, Sirchie Magnetic Silver, failing to produce a profile using any of the methods tested. A link was observed between the DNA extraction chemistry, fingerprint powder property, that is, nonmagnetic, magnetic and aqueous, and the brand of fingerprint powder. Overall, the DNA IQ™ method was favorable for nonmagnetic fingerprint powders, while magnetic fingerprint powders produced more DNA profiles when extracted with the PrepFiler™ chemistry. This study highlights the importance of screening DNA extraction chemistries for the type of fingerprint powder used, as there is not a single DNA extraction method that suits all fingerprint powder brands and properties.

Efficiency of Casework Direct Kit for extraction of touch DNA samples obtained from cars steering wheels

Analysis of STR profiles obtained from touch DNA has been very useful to the elucidation of crimes. Extraction method may be determinant for the recovery of genetic material collected from different surfaces. Vehicle theft is one of the most common crimes in São Paulo city, Brazil, but collection of biological traces in car steering wheels is not considered, because of the belief that profiles generated won’t be able to identify the thief, only the owner. This study aimed to analyze the efficacy of extraction methods for obtaining DNA profiles in samples collected from steering wheels. Eight criminal acts were simulated with 2 different individuals each (mixture of victim and thief), in duplicate, in order to compare two extraction methods: DNA IQ™ and Casework Direct Kit (both Promega Corporation). Genetic material was collected by double swab method and quantified by Quantifiler™Trio (ThermoFisher Scientific). Amplification was conducted with PowerPlex® Fusion System (Promega). It was possible to obtain STR profiles for all experiments. The mixtures were compared with reference profiles to evaluated how many alleles of each donor were observed. Samples extracted with Casework Direct Kit obtained STR profiles with higher averages of alleles for primary and secondary donors (88.7% and 59.9%, respectively) than those extracted with DNA IQ™ (60.4% and 38.1%, respectively). This could be explained by the differences established in the protocols of both methods, since DNA IQ™ is based on successive washes and can result in loss of DNA, whereas Casework Direct Kit minimizes this problem. We concluded that Casework Direct Kit was more efficient for processing touch DNA samples than DNA IQ™.

Validation of a universal DNA extraction method for human and microbiAL DNA analysis

The study of microbiomes has enormous potential for forensic science because microorganisms are ubiquitous and particular communities of microbes are often associated with specific processes or environments. With recent advances in microbiome science, new opportunities exist for microbiome technologies in forensic science (PMI estimation, location of clandestine graves, soil analysis and personal identification). Before a new technology is accepted by the forensic science, it requires an initial validation phase.The aim of our study was to evaluate if the DNA IQ™ Casework Pro Kit for Maxwell® 16 (Promega) is suitable for microbial DNA extraction, without modifications. Ten bacterial strains were selected and subjected to the GenElute Bacterial Genomic DNA extraction protocol (Sigma-Aldrich) and to the DNA IQ™ Casework Pro Kit for Maxwell® 16 protocol. Extracted DNA was quantified and submitted to NGS analysis on an Ion S5 NGS System. Data were analyzed using the Ion Reporter Software metagenomics workflow. Our work has shown that it is possible to purify both microbial and human DNA using the Promega kit, thus making it possible to analyze both human and microbial DNA from a single trace, a pivotal factor in forensics where the quantities of biological material available are usually very limited.

P53 Protein Evaluation on Spermatozoa DNA in Fertile and Infertile Males.

Introduction and Objectives:Protein p53 role in the spermatogenesis is demonstrated, it guarantees both the appropriate quality and quantity of mature spermatozoa. In this observational study we evaluate the eventual correlation between “corrected” p53 concentration on human spermatozoa DNA and male fertility potential.Materials and Methods:Our work is based on an observational study made of 169 male in a period between March 2012 and February 2017. The entire study group is composed by 208 male partners aged between 26-38 years with ejaculate volume from 0.6 to 5.8 mL and heterogeneous seminal valuation: 86/208 (41,3%) normospermic; 19/208 (9,1%) mild oligospermic; 51/208 (24,5%) moderate oligospermic to; 52/208 (25,1%) with severe oligospermic. The “control” group A includes 39 male partners considered “fertile”, because we did the p53 “corrected” test on their spermatozoa after 28 ± 3,5 days from the positives of their partners pregnancy test (betaHCG> 400 m U/m L). The group B, subdivided in B1, B2 and B3, includes 169 male partners for a observational period of 60 months. This partners don't report previous conceptions, they aren't smokers, don't make use neither of alcohol nor drugs and don't present pathologic varicocele studied with ecoColorDoppler. They are all married or stable cohabitants from a period of 27-39 months and report to have frequent sex without protection with their partners. Determination of p53 procedure: To separate the spermatozoa from seminal fluid we utilized the Differex™ kit System and the DNA IQ™ kit (Promega). For the p53 test we used the direct DuoSet IC kit and quantitative (R&D System). The p53 values were corrected in respect to the spermatozoa concentration expressed in ng/millions of spermatozoa.Results:Group A (39 male) presents “correct” p53 values that vary from 0.35 to 3.20 ng/millions of spermatozoa and group B presents values that vary from 0.68 to 14.53. From group B (48 male) in the observational period we have recorded 21 pregnancies with initial “correct” p53 values that vary from a minimum of 0.84 to a maximum of 3.29. In the subgroup B1 we obtained 8 pregnancies from male partners with a “correct” p53 concentration included between 0.84 to 1.34. In the subgroup B2 we obtained 13 pregnancies from male partners with a “correct” p53 concentration included between 1.66 and 3.29. In the subgroup B3 (121 male) there weren't neither pregnancies nor miscarriages and “correct” p53 values were included between 3.58 and 14.53.Conclusion:The results show that the member of the group A with values of 'corrected' p53 between 0.35 and 3.20 were considered “Fertile”, although in the observational period 3 miscarriages happened for 3 partners. 36 partners on 39 (92,3%) had a p53 concentration inferior to 1.65, this value were considered as the extreme to identify this group. The member of the group B1 had “corrected” p53 concentration that were included in the group. In the group B2 were observe 13 pregnancies, so its member were considered “potentially fertile” In the group B3 (121 male) weren't observe neither pregnancies nor miscarriages, so its member were considered “potentially infertile”. If further studies confirm these data, we will consider the p53 test ELISA inspected in “correct” p53 as a new and accurate marker of the potential of male fertility.

Assessment of impact of DNA extraction methods on analysis of human remain samples on massively parallel sequencing success.

Skeletal remains recovered from missing persons' cases are often exposed to harsh environmental conditions resulting in the DNA being damaged, degraded, and/or the samples containing PCR inhibitors. In this study, the efficacy of common extraction methods was evaluated to remove high levels of PCR inhibitors commonly encountered with human remains, and their downstream compatibility with the two leading sequencing chemistries and platforms for human identification purposes. Blood, hair, and bone samples were spiked with high levels of inhibitors commonly identified in each particular substrate in order to test the efficiency of various DNA extraction methods prior to sequencing. Samples were extracted using three commercial extraction kits (DNA IQ™, DNA Investigator, and PrepFiler® BTA), organic (blood and hair only), and two total demineralization protocols (bone only)). Massively parallel sequencing (MPS) was performed using two different systems: Precision ID chemistry and a custom AmpliSeq™ STR and iiSNP panel on the Ion S5™ System and the ForenSeq DNA Signature Prep Kit on the MiSeq FGx™. The overall results showed that all DNA extraction methods were efficient and are fully compatible with both MPS systems. Key performance indicators such as STR and SNP reportable alleles, read depth, and heterozygote balance were comparable for each extraction method. In samples where CE-based STRs yielded partial profiles (bone), MPS-based STRs generated more complete or full profiles. Moreover, MPS panels contain more STR loci than current CE-based STR kits and also include SNPs, which can further increase the power of discrimination obtained from these samples, making MPS a desirable choice for the forensic analysis of such challenging samples.

Comparison of four DNA extraction methods to extract DNA from cigarette butts collected in Lebanon

Cigarette butts collected from crime scenes represent valuable sources of DNA. However the extraction of the genetic material may deem challenging especially when different contaminants may compromise the integrity, quality, and quantity of DNA obtained. This study aims at comparing four extraction methods (Chelex-100, soaking + Chelex-100, Chelex-100 + PK, and DNA IQ™ System) with the intention of identifying the one with maximal recovery rate and profiling success. DNA was extracted using aforementioned four methods from 70 cigarette butts collected from sites across Lebanon. DNA was quantified by qPCR using TaqMan Quantifiler Kit on an Applied Biosystems 7300 SDS instrument and genotypes were obtained using the PowerPlex® 21 kit on an Applied Biosystems 3130 Genetic Analyser. The findings of this work showed that DNA extraction with Chelex-100 + PK is preferred to the other three methods when seeking both, a high yield and the generation of maximal numbers of full profiles. The Chelex-100 + PK method is simple, cost effective, and therefore suitable for routine cigarette butts case studies.

Comparison of commercial touch DNA extraction systems utilizing the polymerase chain reaction technique

Forensic scientists often generate genetic profiles from old blood stains, seminal stains, and hair, but an individual's genetic profile has been accomplished from surfaces touched by human hands. This “Touch DNA” potentially allows for the sensitive identification of DNA from crime scenes where fingerprints are found. The experiment was designed to determine the sensitivity of DNA isolation from fingerprints placed on glass. The two kits used in this experiment were the Invitrogen ChargeSwitch® Forensic DNA Purification Kit and the Promega DNA IQ™ Kit. The kits provided protocols that claimed to facilitate isolation of genomic DNA from fingerprints. After touching cleaned glassware with varying numbers of fingerprints, DNA isolation was performed using each of the above listed kits. A sterile swab was moistened with sterile water and used to extract deposited material from the glassware. Isolated DNA was amplified with the highly sensitive polymerase chain reaction (PCR) technique and separated by gel electrophoresis. A NanoDrop One was used to obtain DNA concentrations for all touch DNA samples and dilutions. The two kits successfully isolated sufficient DNA from cheek swabs and dilutions, and the Promega DNA IQ™ system was found to be more sensitive to DNA contamination. However, the two commercial kits did not successfully extract a sufficient quantity of Touch DNA from a non-porous surface coated with fingerprints. Future experiments should be performed using Touch DNA samples on different surfaces to determine if DNA isolation efficiency can be improved. The project was supported by the NASA West Virginia Space Grant Consortium.

Forensic genetic analysis of bone remain samples

DNA typing from degraded human remains is still challenging forensic DNA scientists not only in the prospective of DNA purification but also in the interpretation of established DNA profiles and data manipulation, especially in mass fatalities. In this report, we presented DNA typing protocol to investigate many skeletal remains in different degrees of decomposing. In addition, we established the grading system aiming for prior determination of the association between levels of decomposing and overall STR amplification efficacy. A total of 80 bone samples were subjected to DNA isolation using the modified DNA IQ™ System (Promega, USA) for bone extraction following with STR analysis using the AmpFLSTR Identifiler® (Thermo Fisher Scientific, USA). In low destruction group, complete STR profiles were observed as 84.4% whereas partial profiles and non-amplified were found as 9.4% and 6.2%, respectively. Moreover, in medium destruction group, both complete and partial STR profiles were observed as 31.2% while 37.5% of this group was unable to amplify. Nevertheless, we could not purify DNA and were unable to generate STR profile in any sample from the high destroyed bone samples. Compact bones such as femur and humerus have high successful amplification rate superior than loose/spongy bones. Furthermore, costal cartilage could be a designate specimen for DNA isolation in a case of the body that was discovered approximately to 3 days after death which enabled to isolate high quality and quantity of DNA, reduce time and cost, and do not require special tools such as freezer mill.

Organic extraction of bone lysates improves DNA purification with silica beads

In our standard protocol for DNA extraction from skeletal remains of unidentified bodies, bone lysates resulting from decalcification and Proteinase K treatment were purified with the DNA IQ™ Casework Pro Kit for Maxwell®16 automate (Promega, WI). Despite its success in the majority of cases, the DNA purification with paramagnetic silica beads failed in some challenging samples. This failure in DNA recovery was often associated with filter clogging during the required volume reduction of the lysate to enable loading on the automate. Two modifications to the standard method were tested for a more efficient filtering and purification. Adding collagenase to the lysate reduced the filter lead time but did not enhance DNA yield, while organic extraction of the crude lysate solved the filter clogging and resulted in successful DNA purification.The modified method in which a phenol treated lysate was loaded on the automate resulted in successful STR-profiling of the skeletal remains of all 13 unidentified bodies tested, which showed a wide variety in post mortem interval and preservation conditions. The variation in DNA yield between the 28 samples tested showed the importance of bone type selection and multiple sampling in successful STR-profiling of skeletal remains.Despite the disadvantages inherent to phenol, the organic extraction of crude bone lysates enhanced the efficiency of DNA purification with paramagnetic silica beads. The combined method of organic extraction and purification with silica beads resulted in STR-profiling of challenging bone samples.

Compatibility of DNA IQ™, QIAamp® DNA Investigator, and QIAsymphony® DNA Investigator® with various fingerprint treatments.

Latent fingerprint and touch DNA are the two most important contact evidence for individualization in forensic science which provide complementary information that can lead to direct and unequivocal identification of the culprit. In order to retrieve useful information from both fingerprints and DNA, which are usually mingled together, one strategy is to perform fingerprint examination prior to DNA analysis since common DNA sampling technique such as swabbing could disturb or even destroy fingerprint details. Here, we describe the compatibility of three automatic DNA extraction systems, namely, DNA IQ™, QIAamp® DNA Investigator, and QIAsymphony® DNA Investigator®, with respective to the effects of various fingerprint detection techniques. Our results demonstrate that Super Glue fingerprint treatment followed by DNA IQ™ extraction shows better effectiveness in DNA profiling. Aluminum powder dusting offers the least interference to the three DNA extraction systems above. Magnetic powder dusting, on the other hand, strongly impedes DNA recovery. Physical Developer is the most intrusive, which yields profiles with poor quality, including lower peak heights, poor peak height ratios, and poor intra-color balance. In terms of the choice of extraction method, DNA IQ™ system is recommended for sampling after fingerprint treatments, but not the two DNA Investigator systems.

Extraction Strategy for DNA Recovery from Putrefied Teeth and Skull Bone

Forensic samples are commonly exposed to harsh environmental conditions which affect the degree of sample (DNA) preservation and subsequent genetic profiling. The aim of this study was to develop a better strategy for DNA extraction from hard putrefied tissues (Teeth and Skull bone). Jaw (teeth) and the skull samples were collected from the putrefied corpses and the authors were asked to determine if the two specimens belonged to the same body. The DNA was extracted by phenol-chloroform and DNA IQ™ System Kit. The PowerPlex® 16 and the PowerPlex® Y System Kits were used for autosomal STR and Y-STR genotyping, respectively. DNA profiling found evidence in favor of DNA degradation. Phenol-Chloroform extracted-DNA was re-extracted by using DNA IQ ™ System kit and managed to identify 13 autosomal STR loci and 13 Y-STR markers from doubly extracted DNA. In conclusion, the combination of two DNA extraction methods (phenol-chloroform + DNA IQ™) improved the quality of DNA extracted from putrefied teeth and skull bone.