Validation of the InnoXtract™ DNA extraction method for bone, teeth, and rootless hair.

Forensic casework samples often include human hairs, teeth, and bones. Hairs with roots are routinely processed for DNA analysis, while rootless hairs are either not tested or processed using mitochondrial DNA. Bones and teeth are submitted for human remains identifications for missing persons and mass disaster cases. DNA extraction from these low templates and degraded samples is challenging. The new InnoXtract DNA extraction method utilizes magnetic beads that are optimized to bind small DNA fragments, as small as 100 base pairs, to purify high-yield DNA from compromised samples. This validation study evaluates InnoXtract's ability to obtain amplifiable DNA from samples such as rootless hairs and skeletal remains. Studies performed include sensitivity, stability, repeatability, reproducibility, non-probative samples, and comparison to standard organic extractions. Sensitivity studies demonstrate average yield recoveries ranging from 53% to 100% and 73% to 85% for the InnoXtract hair and bone methods, respectively. Studies demonstrate consistent results across a range of sample types, such as insulted and un-insulted bone and teeth, as well as hair shafts from donors of various ages, gender, race, and hair characteristics. The InnoXtract bone method outperformed organic extraction. The method was successfully automated on a MagMAX™ Express-96, with recoveries over 70% relative to the manual version. InnoXtract has the potential as an automated high-throughput, high-yield bone extraction method with 6 h of total extraction time for up to 96 samples. The validation study results demonstrate that the InnoXtract kits produce high-yield and high-quality DNA from compromised bone, teeth, and hair shaft samples.

Development and validation of a novel method "SpermX™" for high throughput differential extraction processing of sexual assault kits (SAKs) for DNA analysis.

The Sperm X method uses a nanotechnology derived polymer membrane that functions as a separation medium to effectively trap sperm cells while enabling efficient flow through of the digested epithelial cell DNA. This specialized membrane enabled development of a method that could significantly increase a forensic laboratory's ability to obtain male sperm fraction DNA profiles. The SpermX device provides a rapid, reproducible procedure that is easy to implement in a single-tube format as well as high-throughput truly automated hands-free workflows. Validation studies, performed using the manual SpermX method, include sensitivity, stability, precision (reproducibility and repeatability), mixtures, and a method comparison to the traditional differential extraction. Sensitivity and method comparison studies demonstrated a wide range of sperm cells, from a high of over 2.78 million cells (9158 ng) to a low of 25 cells (83 pg), can be trapped by the SpermX membrane. Stability studies on various substrates (i.e., carpet, cotton, denim, polyester, and silk) and degraded semen gave the expected male DNA profiles. Data from the same operator and a different operator were consistent with low variance. Mixtures, with ratios ranging from approximately 10:1-18182:1, created to simulate real casework type samples including buccal/semen, vaginal epithelial/semen, and post coital swabs at different time intervals, were tested. A comparison of the SpermX method to the conventional differential extraction method resulted in comparable probative male profile allelic data and associated statistical probabilities. For low level sperm samples, down to 25 sperm cells (83 pg), the SpermX method outperformed the conventional differential extraction with more genotypic information and associated probabilities.

Multiplexed real-time polymerase chain reaction cell-free DNA assay as a potential method to monitor stage IV colorectal cancer.

BACKGROUND: Liquid biopsy is a new area in cancer diagnostics that measures cell-free DNA in plasma from tumor that may serve as a monitoring tool in colorectal cancer patients. METHODS: Multiplexed real-time polymerase chain reaction based on multicopy retro-transposable elements (targeting 80 base pair and 265 base pair sequences and an internal-positive-control) was used to evaluate the ability of cell-free DNA concentration and DNA Integrity Index to discriminate cancer from healthy patients. A cohort of 40 healthy controls and 39 stage IV colorectal patient's plasma were interrogated. The potency of each biomarker was measured by using receiver operating characteristic curves and derived area under the curve measures. RESULTS: Significant differences in cell-free DNA concentration and DNA integrity index were observed between controls and stage IV patients with a limit of detection <0.1 pg/µL. Investigation of the ability of both biomarker candidates to differentiate cancer from healthy patients showed an area under the curve of 0.9891 and 0.9859 for 80 base pair and 265 amplicons respectively and 0.8603 for DNA integrity index-265/80. CONCLUSIONS: After establishing differences in cell-free DNA levels between healthy and treated and untreated stage IV patients, the multiplexed real-time polymerase chain reaction measurements of retro-transposable elements in cancer patient plasma potentially possess the ability to monitor therapy responsiveness in near real time.

Successful nuclear DNA profiling of rootless hair shafts: a novel approach.

Historically, rootless hair shaft samples submitted to a forensic laboratory for DNA analysis are reserved for mitochondrial DNA (mtDNA) analysis due to the presence of highly degraded as well as insufficient amounts of nuclear DNA. Although mtDNA has been very successful in obtaining results from rootless hair, this system has its limitations, namely, it is a lineage marker that cannot differentiate between maternally related genotypes. Given the high incidence of hairs as forensic evidence, there is a need for the use of a nuclear DNA test system capable of producing reliable results for hair shaft forensic evidence. This study reports the utilization of an enhanced DNA extraction methodology for hairs, in combination with a recently developed novel, nuclear DNA typing assay, InnoTyper® 21, to improve the success rate for obtaining informative results from highly compromised, degraded, and trace forensic samples such as rootless hair shafts. The InnoTyper 21 kit is a small amplicon retrotransposon marker typing system compatible with currently used capillary electrophoresis platforms. This system contains 20 Alu element markers, ranging in size from 60 to 125 bp, making the assay highly sensitive for extremely degraded forensic samples and thus enabling recovery of nuclear DNA profiles from samples that would otherwise require mtDNA sequencing. A subset of samples was also tested with the GlobalFiler kit with less success due to the larger amplicon sizes in comparison with InnoTyper 21. Results were variable but very promising, with approximately 40% of the total number of hairs tested producing interpretable nuclear DNA profiles with InnoTyper 21. These results demonstrate the ability of the utilized methodologies to produce nuclear DNA results with high statistical power from rootless hair shafts.

Evaluation of the InnoTyper® 21 genotyping kit in multi-ethnic populations.

We report the findings of the evaluation of the InnoTyper® 21 genotyping kit for the use of human identification (HID) and paternity testing in South Africa. This novel forensic kit evaluates 20 retrotransposable elements (AC4027, MLS26, ALU79712, NBC216, NBC106, RG148, NBC13, AC2265, MLS09, AC1141, TARBP, AC2305, HS4.69, NBC51, ACA1766, NBC120, NBC10, NBC102, SB19.12 and NBC148) and the Amelogenin locus for sex determination. The evaluation of the genotyping performance showed no significant spectral pull-up for peak heights between 100 and 30,000 RFUs. All loci presented biallelic patterns except the triallelic RG148 locus resulting from a variant insertion allele, named RG148I-1, observed exclusively in the Bantu. The InnoTyper® 21 kit was found to be highly discriminatory between the 507 unrelated individuals of the Afrikaaner, Asian Indian, Coloured, amaXhosa and amaZulu groups. The HID parameters: the CPD ranged between 0.99999987 and 0.9999999845, and the CMP between 1.0335×10-7 and 1.5506×10-8. The paternity parameters: the CPI ranged between 0.0202 and 0.3177, and the CPE between 0.9161 and 0.9749. There were no significant signs of deviations from HWE or linkage disequilibrium (LD) after applying a Bonferroni correction. This kit also showed minor levels of population structure which could differentiate between the African and non-African population groups. Finally, in challenging casework with severely degraded biological material, the InnoTyper® 21 genotyping kit was compatible with GlobalFiler® and Investigator DIPplex® to increase the HID parameters.

Development and validation of InnoQuant® HY, a system for quantitation and quality assessment of total human and male DNA using high copy targets.

The development and validation of InnoQuant® HY, a real-time PCR system containing four DNA targets-two RE autosomal targets of different sizes, male specific targets, and an internal positive control target-are described herein. The ratio of the two autosomal targets provides a Degradation Index, or a quantitative value of a sample's degradation state. The male specific targets are multi-copy targets located on the Y chromosome, which provides information about a sample's male DNA composition. The experimental results demonstrate InnoQuant HY as a robust qPCR method producing accurate DNA quantitation results even at low dynamic ranges, with reproducibility among population groups. The system is human specific with low level higher primate cross reactivity and is able to consistently and reproducibly detect sub-picogram concentrations of human and human male DNA. The use of high copy number Alu and SVA (>1000 copies per genome) retrotransposable elements as the two autosomal targets significantly enhances both sensitivity and reproducibility of determination of DNA quantitation as well as DNA degradation in forensic samples. The inclusion of a sensitive multi-copy Y-chromosome specific target provides accurate quantitation of DNA from a male in challenging male-female mixtures (i.e. sexual assault samples). Even in the presence of a large excess of DNA from a female, accurate quantitation was achieved with a male to female ratio of 1:128,000. Population database studies reveal an average Short/Y target ratio of the quantification values across all four populations tested was 1.124±0.282, exhibiting the system's reproducibility across multiple populations. The results from InnoQuant HY provide a tool equipping a forensic analyst with crucial data about a sample's DNA quantitation, male:female ratio, degradation state, and the presence or absence of PCR inhibitors. With the information gained from the InnoQuant HY kit, a more streamlined and efficient workflow can be created that minimizes unnecessary sample processing and retesting while maximizing recovery of probative DNA profiles from challenging biological evidence.

Development and validation of a novel multiplexed DNA analysis system, InnoTyper® 21.

We report here a novel multiplexed DNA analysis system consisting of 20 Alu markers and Amelogenin for analysis of highly degraded forensic biological samples. The key to the success of the system in obtaining results from degraded samples is the primer design yielding small amplicon size (60-125bp) for all 20 markers. The markers included in the InnoTyper® 21 system are bi-allelic, having two possible allelic states (insertion or null) and thus termed INNULs. The markers are short interspersed nuclear elements (SINEs), a category of retrotransposable elements (REs) which are non-coding genomic DNA repeat sequences, or "mobile insertion elements," comprising approximately 40% of the human genome. Alu elements are primate specific SINEs that have reached a copy number in excess of one million in the human genome, which makes these markers highly sensitive and desirable for forensic samples with extremely degraded DNA. Until now however, due to the inherent size difference associated with insertion and no insertion alleles, the use of Alu REs has not been practical for forensic applications. The novel primer design described herein has allowed the development of a multiplexed Alu system yielding fragment sizes amenable to degraded DNA samples, as frequently encountered in missing persons cases or forensic samples such as hair shafts. Although use of Alus in human identity has been studied using single marker amplification and reported before, we report for the first time development and validation of a system with multiplexed RE markers. Studies performed include PCR optimization, species specificity, sensitivity, degradation and inhibition, precision and accuracy, nonprobative samples, mixture, and population database studies. A population study using 592 samples including five populations was performed using InnoTyper 21. The data indicated the random match probability for the combination of these 20 Alu markers was greater than 1 in 3.8 million for the populations studied, indicating the greater statistical power of these autosomal nuclear DNA markers over haplotype systems typically used in such degraded samples. Results demonstrate the system is successful in obtaining results from highly degraded DNA. A sensitivity study performed demonstrated at least 95% recovery of alleles from as low as 50pg of total input DNA, and partial profiles from as low as 25pg. This study has demonstrated that the bi-allelic INNULs in the InnoTyper 21 system provide a sensitivity of detection and a power of discrimination that makes them useful for human identification of extremely degraded samples.

Enthalpic switch-points and temperature dependencies of DNA binding and nucleotide incorporation by Pol I DNA polymerases.

This study examines the relationship between the DNA binding thermodynamics and the enzymatic activity of the Klenow and Klentaq Pol I DNA polymerases from Escherichia coli and Thermus aquaticus. Both polymerases bind DNA with nanomolar affinity at temperatures down to at least 5°C, but have lower than 1% enzymatic activity at these lower temperatures. For both polymerases it is found that the temperature of onset of significant enzymatic activity corresponds with the temperature where the enthalpy of binding (ΔHbinding) crosses zero (TH) and becomes favorable (negative). This TH/activity upshift temperature is 15°C for Klenow and 30°C for Klentaq. The results indicate that a negative free energy of DNA binding alone is not sufficient to proceed to catalysis, but that the enthalpic versus entropic balance of binding may be a modulator of the temperature dependence of enzymatic function. Analysis of the temperature dependence of the catalytic activity of Klentaq polymerase using expanded Eyring theory yields thermodynamic patterns for ΔG(‡), ΔH(‡), and TΔS(‡) that are highly analogous to those commonly observed for direct DNA binding. Eyring analysis also finds a significant ΔCp(‡) of formation of the activated complex, which in turn indicates that the temperature of maximal activity, after which incorporation rate slows with increasing temperature, will correspond with the temperature where the activation enthalpy (ΔH(‡)) switches from positive to negative.

Thermodynamics of the DNA structural selectivity of the Pol I DNA polymerases from Escherichia coli and Thermus aquaticus.

Understanding the thermodynamics of substrate selection by DNA polymerase I is important for characterizing the balance between replication and repair for this enzyme in vivo. Due to their sequence and structural similarities, Klenow and Klentaq, the large fragments of the Pol I DNA polymerases from Escherichia coli and Thermus aquaticus, are considered functional homologs. Klentaq, however, does not have a functional proofreading site. Examination of the DNA binding thermodynamics of Klenow and Klentaq to different DNA structures: single-stranded DNA (ss-DNA), primer-template DNA (pt-DNA), and blunt-end double-stranded DNA (ds-DNA) show that the binding selectivity pattern is similar when examined across a wide range of salt concentration, but can significantly differ at any individual salt concentration. For both proteins, binding of single-stranded DNA shifts from weakest to tightest binding of the three structures as the salt concentration increases. Both Klenow and Klentaq release two to three more ions when binding to pt-DNA and ds-DNA than when binding to ss-DNA. Klenow exhibits significant differences in the Delta C(p) of binding to pt-DNA versus ds-DNA, and a difference in pI for these two complexes, whereas Klentaq does not, suggesting that Klenow and Klentaq discriminate between these two structures differently. Taken together, the data suggest that the two polymerases bind ds-DNA very differently, but that both bind pt-DNA and ss-DNA similarly, despite the absence of a proofreading site in Klentaq.