An 18 Multi-InDels panel for analysis of highly degraded forensic biological samples

DNA genotyping from trace and highly degraded biological samples is one of the most significant challenges of forensic DNA identification. There is a lack of simple and effective methods for genotyping highly degraded samples. In this study, a multiple loci insertion/deletion polymorphisms (Multi-InDels) panel was designed for detecting 18 autosomal Multi-InDels through capillary electrophoresis (CE) with amplicon sizes no longer than 125 bp. Studies of sensitivity, degradation, and species specificity were performed and a population study was carried out using 192 samples from Han populations in Hunan province in the south of China. The combined random match probability (CMP) of these 18 Multi-InDels was 3.23 × 10^–12 and the cumulative probability of exclusion (CPE) was 0.9989, suggesting this panel could be used independently for human identification and could provide efficient supporting information for parentage testing. Complete profiles were obtained from as low as 62.5 pg of total input DNA after increasing the number of PCR cycles. Moreover, all alleles were detected from artificially highly degraded DNA after 80 min of boiling water bath treatment. This 18 Multi-InDels panel is simple, fast, and effective for the forensic analysis of highly degraded DNA.     

The application of magnetic bead hybridization for the recovery and STR amplification of degraded and inhibited forensic DNA

A common problem in the analysis of forensic DNA evidence is the presence of environmentally degraded and inhibited DNA. Such samples produce a variety of interpretational problems such as allele imbalance, allele dropout and sequence specific inhibition. In an attempt to develop methods to enhance the recovery of this type of evidence, magnetic bead hybridization has been applied to extract and preconcentrate DNA sequences containing short tandem repeat (STR) alleles of interest. In this work, genomic DNA was fragmented by heating, and sequences associated with STR alleles were selectively hybridized to allele-specific biotinylated probes. Each particular biotinylated probe-DNA complex was bound to streptavidin-coated magnetic beads using enabling enrichment of target DNA sequences. Experiments conducted using degraded DNA samples, as well as samples containing a large concentration of inhibitory substances, showed good specificity and recovery of missing alleles. Based on the favorable results obtained with these specific probes, this method should prove useful as a tool to improve the recovery of alleles from degraded and inhibited DNA samples.     

Improved STR analysis of degraded DNA from human skeletal remains through in-house MPS-STR panel

DNA analysis of degraded samples and low-copy number DNA derived from skeletal remains, one of the most challenging forensic tasks, is common in disaster victim identification and genetic analysis of historical materials. Massively parallel sequencing (MPS) is a useful technique for STR analysis that enables the sequencing of smaller amplicons compared with conventional capillary electrophoresis (CE), which is valuable for the analysis of degraded DNA. In this study, 92 samples of human skeletal remains (70+ years postmortem) were tested using an in-house MPS-STR system designed for the analysis of degraded DNA. Multiple intrinsic factors of DNA from skeletal remains that affect STR typing were assessed. The recovery of STR alleles was influenced more by DNA input amount for amplification rather than DNA degradation, which may be attributed from the high quantity and quality of libraries prepared for MPS run. In addition, the higher success rate of STR typing was achieved using the MPS-STR system compared with a commercial CE-STR system by providing smaller sized fragments for amplification. The results can provide constructive information for the analysis of degraded sample, and this MPS-STR system will contribute in forensic application with regard to skeletal remain sample investigation.     

Characterization of sequence variation at 30 autosomal STRs in Chinese Han and Tibetan populations

Massively parallel sequencing (MPS) technologies have the ability to reveal sequence variations within STR alleles as well as their nominal allele lengths, which have traditionally been detected by CE instruments. Recently, Thermo Fisher Scientific has updated the MPS-STR panel, named the Precision ID GlobalFiler next-generation sequencing (NGS) STR Panel version 2, with primers redesigned to add two pentanucleotide tandem repeat loci and profile interpretation supported by the Converge software. Using the Ion Chef System, the Ion S5XL System, and the Converge software, genetic variations were characterized within STR repeat and flanking regions of 30 autosomal STR markers in 115 unrelated individuals from two Chinese population groups (58 Tibetans and 57 Hans). Nineteen STRs demonstrated a relative increase in diversity with the variant sequence alleles compared with those of traditional nominal length alleles. In total, 390 alleles were identified by their sequences compared with 258 alleles that were identified by length. Of these 92 sequence variants found within the STR repeat regions, 40 variants were located in STR flanking regions. Additionally, the agreement of the results with CE data was evaluated, as was the ability of this new MPS panel to analyze case-type (11 samples) and artificially degraded samples (seven samples in triplicate). The results generated from this study illustrate that extensive sequence variation exists in commonly used STR markers in the selected population samples and indicate that this NGS STR panel has the potential to be used as an effective tool for human forensics.     

Determination of rosuvastatin in the presence of its degradation products by a stability-indicating LC method

A forced degradation study was successfully applied for the development of a stability-indicating assay method for determination of rosuvastatin Ca in the presence of its degradation products. The method was developed and optimized by analyzing the forcefully degraded samples. Degradation of the drug was done at various pH values. Moreover, the drug was degraded under oxidative, photolytic, and thermal stress conditions. Mass balance between assay values of degraded samples and generated impurities was found to be satisfactory. The proposed method was able to resolve all of the possible degradation products formed during the stress study. The developed method was successfully applied for an accelerated stability study of the tablet formulation. The major impurities generated during the accelerated stability study of the tablet formulation were matches with those of the forced degradation study. The developed method was validated for determination of rosuvastatin Ca, and the method was found to be equally applicable to study the impurities formed during routine and forced degradation of rosuvastatin Ca.     

CE combined with rolling circle amplification for sensitive DNA detection

Here we describe an assay which combines CE with rolling circle amplification (RCA) for sensitive DNA detection and quantification. RCA is an isothermal DNA replication technique that generates a long ssDNA with tandem repeats. It requires simpler temperature control in reaction and offers higher sequence specificity and greater quantitation capability compared to other amplification technologies. In this study, RCA amplified the DNA target via a circular template, and the product was digested into monomers for CE analysis. Less than 2 fmol of the DNA target could easily be detected using this RCA-CE assay and the assay has a dynamic range of two orders of magnitudes. Moreover, simultaneous detection of both the target DNA and the internal standard was achieved by designing two padlock probes with different sizes, which could significantly improve the quantification accuracy. The RCA-CE assay is easy to perform, readily adaptable for detection of multiple targets because of the high resolution power of CE, and is compatible with other applications employing RCA as a signal amplification tool. Additionally, this assay can be used with a capillary array system to perform sensitive, high-throughput genetic screening.     

Development of internal amplification controls for DNA profiling with the AmpFℓSTR(®) SGM Plus(®) kit

DNA extracted from forensic samples can be degraded and also contain co‐extracted contaminants that inhibit PCR. The effects of DNA degradation and PCR inhibition are often indistinguishable when examining a DNA profile. Two internal amplification controls (IACs) were developed to improve quality control of PCR using the AmpF?STR^® SGM Plus^® kit. The co‐amplification of these controls with DNA samples was used to monitor amplification efficiency and detect PCR inhibitors. IAC fragments of 90 and 410 bp (IAC₉₀ and IAC₄₁₀) were generated from the plasmid pBR322 using tailed primers and then amplified with ROX‐labelled primers. Co‐amplification of IAC₉₀ and IAC₄₁₀ was performed with varying amounts of template DNA, degraded DNA and DNA contaminated with humic acid, heme and indigo dye. Both IAC₉₀ and IAC₄₁₀ were successfully amplified with human DNA without significantly affecting the quality of the DNA profile, even with DNA amounts lower than 0.5 ng. In the presence of inhibitors, the IAC₉₀ signal was still present after all human DNA loci fail to amplify; in contrast, the IAC₄₁₀ signal was reduced or absent at low levels of inhibition. Amplification of the two IACs provided an internal PCR control and allowed partial profiles caused by inhibition to be distinguished from degraded DNA profiles.     

Influence of DNA extraction methods, PCR inhibitors and quantification methods on real-time PCR assay of biotechnology-derived traits

Biotechnology-derived varieties of canola, cotton, corn and soybean are being grown in the USA, Canada and other predominantly grain exporting countries. Although the amount of farmland devoted to production of biotechnology-derived crops continues to increase, lingering concerns that unintended consequences may occur provide the EU and most grain-importing countries with justification to regulate these crops. Legislation in the EU requires traceability of grains/oilseeds, food and feed products, and labelling, when a threshold level of 0.9% w/w of genetically engineered trait is demonstrated to be present in an analytical sample. The GE content is routinely determined by quantitative PCR (qPCR) and plant genomic DNA provides the template for the initial steps in this process. A plethora of DNA extraction methods exist for qPCR applications. Implementing standardized methods for detection of genetically engineered traits is necessary to facilitate grain marketing. The International Organization for Standardization draft standard 21571 identifies detergent-based methods and commercially available kits that are widely used for DNA extraction, but also indicates that adaptations may be necessary depending upon the sample matrix. This review assesses advantages and disadvantages of various commercially available DNA extraction kits, as well as modifications to published cetyltrimethylammonium bromide methods. Inhibitors are a major obstacle for efficient amplification in qPCR. The types of PCR inhibitors and techniques to minimize inhibition are discussed. Finally, accurate quantification of DNA for applications in qPCR is not trivial. Many confounders contribute to differences in analytical measurements when a particular DNA quantification method is applied and different methods do not always provide concordant results on the same DNA sample. How these differences impact measurement uncertainty in qPCR is considered.     

A comparison between petrous bone and tooth,femur and tibia DNA analysis from degraded skeletal remains

Skeletal remains are the only biological material that remains after long periods; however, environmental conditions such as temperature, humidity, and pH affect DNA preservation, turning skeletal remains into a challenging sample for DNA laboratories. Sample selection is a key factor, and femur and tooth have been traditionally recommended as the best substrate of genetic material. Recently, petrous bone (cochlear area) has been suggested as a better option due to its DNA yield. This research aims to evaluate the efficiency of petrous bone compared to other cranium samples (tooth) and postcranial long bones (femur and tibia). A total amount of 88 samples were selected from 38 different individuals. The samples were extracted by using an organic extraction protocol, DNA quantification by Quantifiler Trio kit and amplified with GlobalFiler kit. Results show that petrous bone outperforms other bone remains in quantification data, yielding 15–30 times more DNA than the others. DNA profile data presented likeness between petrous bone and tooth regarding detected alleles; however, the amount of DNA extracted in petrous bones allowed us to obtain more informative DNA profiles with superior quality. In conclusion, petrous bone or teeth sampling is recommended if DNA typing is going to be performed with environmentally degraded skeletal remains.     

Development and validation of a custom panel including 183 Y-SNPs for Chinese Y-chromosomal haplogroups dissection using a MALDI-TOF MS system

Y-chromosome SNP haplogroups exhibit geographic structuring in many populations around the world. Therefore, Y-chromosome haplogroups have been widely used to infer paternal biogeographical ancestry and high-resolution paternal lineage classification. In the present study, we designed a customized panel containing 183 Y-SNPs based on previous studies and evaluated the genotyping performance and repeatability, concordance, sensitivity, and ability of analysing case-type samples using a MALDI-TOF MS platform. The average call rate for duplicate typing of any one SNP in the panel was 97.0%. In the concordance and accuracy study, the results of haplogroup designation obtained from MALDI-TOF MS platform were fully consistent with those obtained from the next-generation sequencing (NGS) platform. The optimal amount of template DNA in the PCR seemed to be 10 ng. However, if less DNA (≥156.25 pg) was available, it was still possible to obtain meaningful haplogroup information. For the application part, this panel could be applied for the detection of blood, semen, and buccal swabs samples. Particularly, blood stain on FTA card samples could be dissected by direct PCR amplification on the MALDI-TOF MS platform. Besides, 371 unrelated male individuals from four Chinese ethnic groups (Han, Hui, Mongolian, and Kazak) were detected using this panel. Total 78 terminal haplogroups were found and the haplogroup diversity was 0.933576. The results demonstrate that this panel could be an accurate, fast, and cost-effective method for database construction where the amount of sample material is less of a concern and when the cost of the assay is taken into consideration.     

Accuracy in naturally occurring anabolic steroid assays in cattle and first approach to quality control in Italy

This study assessed the accuracy of 16 commercial and three self-produced kits and drew the basis for using an external quality control (EQC) system. The commercial kits were mainly developed for blood sex steroid determination in humans but also have been used in cattle. Parallelism, recovery and precision tests were performed for progesterone (P4), testosterone (T) and oestradiol (E2) assays. Moreover, anonymous QC samples were sent to be analysed to some Italian laboratories. All kits showed a fair degree of parallelism (P < 0.01), even though 2/7 kits for T and 1/6 kits for E2 determination showed a regression coefficient (r2) lower than 0.98. For P4, an acceptable range of accuracy was achieved in the recovery test only by 1/6 kits; two kits showed fair or great overestimation and two kits considerable underestimation. For T, an acceptable range of accuracy was achieved only by 1/7 kits. For E2, 4/6 kits presented a variable degree of underestimation and two kits showed great overestimation. In the intra-assay precision test quite good repeatability was achieved only using samples with high hormone concentrations. While assaying samples with low concentrations we found a number of RSD > 10%. Moreover, the laboratories participating in the EQC produced statistically different (P < 0.05) results, particularly for high and medium concentrations. In conclusion, the use of commercial kits for screening naturally occurring sex steroid concentrations in cattle blood, in the case of suspected illegal treatments, requires preventive validation procedures and the development of an opportune EQC system.     

QUANTITATION OF PYRIMIDINE DIMERS BY IMMUNOSLOT BLOT FOLLOWING SUBLETHAL UV-IRRADIATION OF HUMAN CELLS

An immunoslot blot assay was developed to detect pyrimidine dimers induced in DNA by sublethal doses of UV (254 nm) radiation. Using this assay, one dimer could be detected in 10 megabase DNA using 200 ng or 0.5 megabase DNA using 20 ng irradiated DNA. The level of detection, as measured by dimer specific antibody binding, was proportional to the dose of UV and amount of irradiated DNA used. The repair of pyrimidine dimers was measured in human skin fibroblastic cells in culture following exposure to 0.5 to 5 J m^-2 of 254 nm UV radiation. The half-life of repair was approximately 24, 7 and 6 h in cells exposed to 0.5, 2 and 5 J m^-2 UV radiation, respectively. This immunological approach utilizing irradiated DNA immobilized to nitrocellulose should allow the direct quantitation of dimers following very low levels of irradiation in small biological samples and isolated gene fragments.     

Optimized suppression of adducts in polymerase chain reaction products for semi-quantitative SNP genotyping by liquid chromatography-mass spectrometry

While electrospray ionization mass spectrometry has shown great potential for the identification of genotypes in DNA sequences amplified by polymerase chain reaction (PCR), the quantitative determination of allele frequencies remains challenging because of the presence of cationic adducts in the mass spectra which severely impairs accuracy of quantitation. We report here on the elaboration of an optimized desalting protocol for ion-pair reversed-phase high-performance liquid chromatography-electrospray ionization mass spectrometry (ICEMS) of PCR amplicons which facilitates the genotyping of single nucleotide polymorphisms (SNPs). Chromatographic purification at temperatures between 50 and 70 degrees C using monolithic reversed-phase columns and acetonitrile gradients in aqueous, 20-30 mmol/l butyldimethylammonium bicarbonate enabled the mass spectrometric analysis of nucleic acid solutions containing up to 1.7 mol/l sodium chloride. A further improvement in removal of metal cations was achieved upon the addition of 5-10 mmol/l ethylenediaminetetraacetic acid (EDTA) to the sample solution prior to liquid chromatography. ICEMS was used for the semi-quantitative genotyping of SNPs amplified from the tetraploid genome of potato cultivars. Using a quadrupole ion trap mass spectrometer, allele frequencies were determined with an accuracy of 2-9% by measuring the relative intensities of the signals corresponding to the molecular mass of each of the alleles in the deconvoluted mass spectra. ICEMS results correlated well with those obtained by pyrosequencing, single nucleotide primer extension, and conventional dideoxy sequencing.     

Quantitative analysis of volatiles from solid matrices of vegetable origin by high concentration capacity headspace techniques: determination of furan in roasted coffee

The study compares standard addition (SA), stable isotope dilution assay (SIDA) and multiple headspace extraction (MHE) as methods to quantify furan and 2-methyl-furan in roasted coffee with HS-SPME-GC-MS, using CAR-PDMS as fibre coating, d(4)-furan as internal standard and in-fibre internal standardization with n-undecane to check the fibre reliability. The results on about 150 samples calculated with the three quantitation approaches were all very satisfactory, with coefficient of variation (CV) versus the U.S. Food and Drug Administration (FDA) method, taken as reference, almost always below the arbitrarily-fixed limit of 15%. Furan was detected in the 1-5 ppm range, 2-methyl-furan in the 4-20 ppm range. Moreover, experimental exponential slopes (Q) and linearity (r) of both furan and 2-methyl-furan MHE regression equation on 50 samples were very similar thus making possible to use the same average Q value for all samples of the investigated set and their quantitation with a single determination. This makes this approach very rapid and competitive in-time with SA and SIDA. A non-separative method (HS-SPME-MS) was also developed in view of possible application on-line monitoring of furan and 2-methyl-furan in a pilot-plant with the aim of optimizing the roasting process to reduce these compounds to a minimum. Sampling times of 20 and 5 min were tested, the latter enabling total analysis time to be reduced to about 9 min. The results on 105 samples with both SIDA and MHE approaches were again highly satisfactory most of the samples giving a CV% versus the conventional methods below 20%. In this case too average Q values for both furan and 2-methyl-furan were used for MHE. The separative method presented very good repeatability (RSD% always below 10%) and intermediate precision over three months (RSD% always below 15%); performance were similar for the non-separative method, with repeatability (RSD%) always below 12% and intermediate precision over three months (RSD%) always below 15%. The sensitivity of both separative and non-separative methods was also very good, LOD and LOQ being in the ppb range for both furan and 2-methyl-furan, i.e. well below the amounts present in the roasted coffee samples.     

A simple and rapid method for HLA-DQA1 genotyping by polymerase chain reaction-single strand conformation polymorphism and restriction enzyme cleavage analysis.

A simple and rapid method for identification of alleles at the human leucocyte antigen (HLA)-DQA1 locus is described. The polymorphic second exon of the HLA-DQA1 locus was amplified by the polymerase chain reaction (PCR) method. The amplified DNA was analyzed by single-strand conformation polymorphism (SSCP) and restriction enzyme cleavage assay. Using this method, the eight known DQA1 alleles could be distinguished from each other. This paper suggests that the method can be used for quick genotyping of DQA1 alleles, but detecting point mutations at various positions in a fragment as well as new HLA-DQA1 genotypes should also be possible.     

Rapid determination of fosetyl-aluminum residues in lettuce by liquid chromatography/electrospray tandem mass spectrometry

This paper describes a new method for the sensitive and selective determination of fosetyl-aluminum (Al) residues in vegetable samples. The method involves extraction with water by using a high-speed blender and subsequent injection of the 5-fold diluted extract into the liquid chromatograph. Fosetyl-Al is determined by liquid chromatography with electrospray tandem mass spectrometry after the addition of tetrabutylammonium acetate as the ion-pairing reagent. The method has been used to assay lettuce samples spiked at 2 and 0.2 mg/kg. Recoveries were satisfactory, with mean values of 98 and 106%, respectively, and relative standard deviations were < 10%. The limit of quantitation was 0.2 mg/kg, and the limit of detection was as low as 0.05 mg/kg. Matrix-matched calibration was used for quantitation, and the addition of an internal standard improved repeatability. The developed method allows the accurate and rapid determination of low levels of fosetyl-Al residues in lettuce with very little sample handling and good sensitivity; it was shown to be robust by the analysis of almost 100 samples.     

Disposable Stochastic Dot Sensors for the Assay of Ascorbic Acid in Pharmaceutical Samples,Beverages, and Biological Fluids

Eight disposable stochastic dot sensors based on porphyrins and modified diamond or carbon pastes were employed for the assay of ascorbic acid in pharmaceutical, beverages, and biological samples. The advantage of using such sensors for the assay of ascorbic acid is the possibility of qualitative and quantitative assay of ascorbic acid in one run of the experiment. The covered linear concentration range for these sensors was between 10^?14 and 10^?5 mol/L with high sensitivities. The proposed dot sensors were used reliably (RSD < 1%) for the assay of ascorbic acid from different samples for more than 6 months, with a recovery higher than 92.00%.     

Development and validation of a forensic multiplex InDel assay: The AGCU InDel 60 kit

Marker sets based on insertion/deletion polymorphisms (InDels) combine the characteristics of both short tandem repeats (STRs) and single nucleotide polymorphisms and have served as effective complementary or stand-alone systems for human identification in forensics. We developed a novel multiplex amplification detection system, designated the AGCU InDel 60 kit, containing 57 autosomal InDels, 2 Y-chromosomal InDels, and the amelogenin locus and validated the kit in a series of studies, which included tests of the PCR conditions; tests for sensitivity, species specificity, reproducibility, stability, and mock case samples; degradation studies; and a population study. The results indicated that the AGCU InDel 60 kit was accurate, specific, reproducible, stable, and robust. Complete DNA profiles were obtained even with 125 pg of human DNA. In tests of artificially degraded samples, we found that the number of alleles detected by the validated kit was considerably greater than that detected by the STR-based AGCU 21+1 kit, even as the degree of degradation increased. Additionally, 564 unrelated individuals from three Han groups were investigated using this novel system, and the values of combined power of discrimination and combined power of exclusion were not less than 1–4.9026 × 10⁻²⁴ and 1–3.1123 × 10⁻⁵, respectively. Thus, the results indicated that the novel kit was more powerful than the previous version of the InDel kit (the AGCU InDel 50 kit). Our results suggest that the AGCU InDel 60 kit can serve as an efficient tool for human forensics and a supplementary kit for population genetics research.     

In Vitro Selection of a DNA Aptamer Targeting Degraded Protein Fragments for Biosensing

Protein biomarkers often exist as degradation fragments in biological samples, and affinity agents derived using a purified protein may not recognize them, limiting their value for clinical diagnosis. Herein, we present a method to overcome this issue, by selecting aptamers against a degraded form of the toxin B protein, which is a marker for diagnosing toxigenic Clostridium difficile infections. This approach has led to isolation of a DNA aptamer that recognizes degraded toxin B, fresh toxin B, and toxin B spiked into human stool samples. DNA aptamers selected using intact recombinant toxin B failed to recognize degraded toxin B, which is the form present in stored stool samples. Using this new aptamer, we produced a simple paper-based analytical device for colorimetric detection of toxin B in stool samples, or in the NAP1 strain of Clostridium difficile. The combined aptamer-selection and paper-sensing strategy can expand the practical utility of DNA aptamers in clinical diagnosis.     

Real-time polymerase chain reaction detection of cauliflower mosaic virus to complement the 35S screening assay for genetically modified organisms

Labeling of genetically modified organisms (GMOs) is now in place in many countries, including the European Union, in order to guarantee the consumer's choice between GM and non-GM products. Screening of samples is performed by polymerase chain reaction (PCR) amplification of regulatory sequences frequently introduced into genetically modified plants. Primers for the 35S promoter from Cauliflower mosaic virus (CaMV) are those most frequently used. In virus-infected plants or in samples contaminated with plant material carrying the virus, false-positive results can consequently occur. A system for real-time PCR using a TaqMan minor groove binder probe was designed that allows recognition of virus coat protein in the sample, thus allowing differentiation between transgenic and virus-infected samples. We measured the efficiency of PCR amplification, limits of detection and quantification, range of linearity, and repeatability of the assay in order to assess the applicability of the assay for routine analysis. The specificity of the detection system was tested on various virus isolates and plant species. All 8 CaMV isolates were successfully amplified using the designed system. No cross-reactivity was detected with DNA from 3 isolates of the closely related Carnation etched ring virus. Primers do not amplify plant DNA from available genetically modified maize and soybean lines or from different species of Brassicaceae or Solanaceae that are natural hosts for CaMV. We evaluated the assay for different food matrixes by spiking CaMV DNA into DNA from food samples and have successfully amplified CaMV from all samples. The assay was tested on rapeseed samples from routine GMO testing that were positive in the 35S screening assay, and the presence of the virus was confirmed.