Subsequent Short Tandem Repeat Research Articles

Optimized Recovery of DNA and Subsequent Short Tandem Repeat Profiling of Different Tissues Sampled from Embalmed Human Cadavers

Abstract Introduction: Storage of specimens sampled from human remains for pathological testing, embalming for burial purposes, and for human identification often requires formalin fixation and/or paraffin embedding. Current knowledge in molecular biology techniques and forensic DNA analysis makes it possible to optimize the extraction of amplifiable DNA from formalin-fixed tissues by improving the pre-treatment, optimizing the digestion condition of proteinase K, simplifying the extraction protocol and purifying the extracted DNA with optimized volumes of alcohol. Aim: This research sought to extract amplifiable DNA from thirteen brain, bone marrow and cartilage samples from four formalin embalmed human cadavers. Materials and Methods: Brain, cartilage and bone marrow samples were taken from four different cadavers at autopsy at the Ghana Police Hospital mortuary in Accra, Ghana sixty-two days after embalming. An optimized preparation and DNA extraction protocol was carried out on all the samples. Brain samples were also taken from a non-formalin treated fifth cadaver of known STR profile, and standard DNA extraction performed to serve as positive control. Results: Our optimized protocol yielded detectable quantities of DNA from the samples when quantified with the 7500 Real-Time PCR equipment. The extracted DNA also yielded full STR profiles with varying peak heights for forensic identification purposes. The measured degradation indexes of the DNA samples were greater than 1.0, with peak heights of generated STR profiles above the limits of detection of the 3500 genetic analyzer. Conclusion: Our current study demonstrated an optimized method of DNA extraction from tissues (brain, cartilage and bone marrow) sampled from formalin embalmed human cadavers. The optimized protocol reduced the concentration of formalin fixation residues in extracted DNA from formalin-fixed tissues, thereby improving the amplification efficiency for STR profiling. Brain, bone marrow and cartilages can be a good source of DNA from embalmed and degraded human remains, though for skeletonized human remains together with teeth and long bones.

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

The quantification of human genomic DNA is a necessary first step in the DNA casework sample analysis workflow. DNA quantification determines optimal sample input amounts for subsequent STR (short tandem repeat) genotyping procedures, as well as being a useful screening tool to identify samples most likely to provide probative genotypic evidence. To better mesh with the capabilities of newest-generation STR analysis assays, the Quantifiler® HP and Quantifiler® Trio DNA Quantification Kits were designed for greater detection sensitivity and more robust performance with samples that contain PCR inhibitors or degraded DNA. The new DNA quantification kits use multiplex TaqMan® assay-based fluorescent probe technology to simultaneously quantify up to three human genomic targets, allowing samples to be assessed for total human DNA, male contributor (i.e., Y-chromosome) DNA, as well as a determination of DNA degradation state. The Quantifiler HP and Trio Kits use multiple-copy loci to allow for significantly improved sensitivity compared to earlier-generation kits that employ single-copy target loci. The kits’ improved performance provides better predictive ability for results with downstream, newest-generation STR assays, and their shortened time-to-result allows more efficient integration into the forensic casework analysis workflow.

Quantification of mixed chimerism allows early therapeutic interventions.

Hematopoietic stem cell transplantation is the curative option for patients with myelodysplastic syndrome; however, it requires a long post-transplantation follow-up. A 53-year-old woman with a diagnosis of myelodysplastic syndrome underwent related donor allogeneic hematopoietic stem cell transplantation in July 2006. Three months after transplantation, a comparative short tandem repeat analysis between donor and recipient revealed full chimerism, indicating complete, healthy bone marrow reconstitution. Three years and ten months after hematopoietic stem cell transplantation, the patient developed leukopenia and thrombocytopenia. Another short tandem repeat analysis was carried out which showed mixed chimerism (52.62%), indicating relapsed disease. A donor lymphocyte infusion was administered. The purpose of donor lymphocyte infusion is to induce a graft-versus-leukemia effect; in fact, this donor's lymphocyte infusion induced full chimerism. Successive short tandem repeat analyses were performed as part of post-transplantation follow-up, and in July 2010, one such analysis again showed mixed chimerism (64.25%). Based on this finding, a second donor lymphocyte infusion was administered, but failed to eradicate the disease. In September 2011, the patient presented with relapsed disease, and a second related donor allogeneic hematopoietic stem cell transplantation was performed. Subsequent short tandem repeat analyses revealed full chimerism, indicating complete bone marrow reconstitution. We conclude that quantitative detection of mixed chimerism is an important diagnostic tool that can guide early therapeutic intervention.

Effect of Dactyloscopic Powders on DNA Profiling From Enhanced Fingerprints

We conducted a study on the effect of fingerprint enhancement methods on subsequent short tandem repeat profiling. First, we performed a study typing blood traces deposited on 5 different surfaces, treated with 8 types of dactyloscopic powders. Three different DNA extraction methods were used. Subsequently, we analyzed latent fingerprints on the same 5 surfaces enhanced with the 8 different powders used in the first part of the study. This study has demonstrated that DNA profiling can be performed on fingerprints left on different substrates, and the substrate will affect the amount of DNA that can be recovered for DNA typing. In the first phase of the study, a profile was obtained in 92% of the 120 samples analyzed; in the second part, in 55% of the 80 samples analyzed, we obtained a profile complete in 32.5% of the cases. From the results obtained, it seems that the powders used in latent fingerprints enhancement, rather than having a direct inhibitory effect on extraction and amplification of DNA, may cause partial degradation of DNA, reducing the efficiency of amplification reaction. It should not be forgotten that these results were obtained under laboratory conditions, and in real caseworks, there may still be different problems involved.

指紋検出に用いる粉末および転写シートが STR 型検査に及ぼす影響

We examined whether powders and transfer sheets for detecting latent fingerprints affect DNA extraction and subsequent short tandem repeat (STR) typing adversely. To examine the powders, saliva and blood samples were smeared and dried on glass slides, and then adhered with aluminum powder, black powder, magnetic powder (NBS), magnetic powder (Sirche), SP black and indigo. To examine transfer sheets, saliva and blood samples were smeared and dried on gelatin sheets, JP sheet and Lifter. DNA was extracted from the powder-adhered samples or the samples on the sheets, quantified by a real-time PCR assay, and the STR typing was performed using the Identifiler kit. The aluminum powder and the SP black affected the storage of the DNA solution, and required a centrifugation step to remove the powders, or required TE−4 buffer for DNA elution instead of water. The magnetic powder (NBS) made the DNA extraction impossible, when an excessive amount of the powder was adhered. However, the DNA extraction was possible, when most of the powder was removed with a magnetic brush. The black powder, the magnetic powder (Sirche) and the indigo gave enough DNA concentration and full STR profiles. All the transfer sheets did not give adverse effects. As simulated casework samples, 20 fingerprints deposited on a glass slide in two different ways were enhanced with the aluminum powder and transferred to the gelatin sheets. DNA was extracted from the gelatin sheets, and the residual portion on the glass slide. The DNA concentration ≥0.01 ng/μl was obtained from 2 samples for the residual portion of 20 fingerprints deposited 2 hours after washing hands, and 7 samples for that of 20 samples deposited after touching forehead, cheeks and neck. The STR typing was performed from the samples ≥0.01 ng/μl. Full STR profiles were obtained from 6 samples (≥0.024 ng/μl) and partial STR profiles from 3 samples (0.10-0.12 ng/μl) when the residual portion used.

A bone sample cleaning method using trypsin for the isolation of DNA

Cleaning the surface of bone samples is a necessary step to remove contaminants prior to isolating DNA for forensic DNA analysis. In this study, a simple trypsin method for cleaning bone samples prior to DNA isolation was developed. Cleaning the surface of human bone samples was achieved by the application of trypsin solution. Light microscopy and scanning electron microscopy results indicated that trypsin treatment was effective in removing the outer surface of bone samples. The yield of DNA isolated from trypsin-treated bone samples was sufficient for subsequent short tandem repeat (STR) analysis. STR analysis revealed no adverse effect on the DNA profile after the trypsin treatment. The data suggest that this trypsin method can potentially be an alternative cleaning method to mechanical cleaning methods.

The Effect of the Methods for Detecting Blood Fingerprints on STR Typing

In Japan, blood fingerprints found at crime scenes are photographed and transferred to a Pap or BB sheet. The transferred fingerprints are, if necessary, enhanced by a solution of tetramethylbenzidine (TMB) or leucomalachite green (LMG). The effects of TMB or LMG on short tandem repeat (STR) typing have been studied. However, the total effects of blood fingerprints detection methods on subsequent STR typing have not been investigated enough. In this study, 3 μl of blood and 20 μl of saliva samples smeared on glass slides were transferred to a Pap or BB sheet, transferred to a BB sheet and applied with TMG or LMG solution, and only sprayed with TMG or LMG solution. DNA was extracted by an EZ-1 DNA Investigator kit or by a QIAamp DNA Investigator kit. The quantity of DNA was measured by real-time PCR assay and STR typing was performed. The Pap sheet-transferred portions of the samples showed no STR profiles, regardless of the DNA extraction kits. On the other hand, the BB sheet-transferred portions showed full STR profiles in all the samples. The residual portions on the glass slide that had not been transferred to Pap or BB sheets, showed full STR profiles in most of the samples. The QIAamp kit had a tendency to provide a higher quantity of DNA than the EZ-1 kit. TMB hardly or slightly reduced the quantity of DNA, but full STR profiles were still obtained. The application of LMG to the BB-sheet transferred portions drastically reduced the quantity of DNA, and affected STR typing adversely. The spraying of LMG on the samples on glass slides slightly reduced the quantity of DNA, but gave full STR profiles. It was recommended that STR typing be performed from residual portions of samples that have not been transferred to Pap or BB sheets.

Recipient-Derived Neoangiogenesis of Arterioles and Lymphatics in Quilty Lesions of Cardiac Allografts

The contribution of extracardiac cells to tissue turnover in heart allografts has recently been demonstrated. Complex subendocardial infiltrates, known as Quilty lesions, are frequently observed in cardiac allografts. The origin of the different cellular components of Quilty lesions is not known. Different constituents of these lymphonodular infiltrates were analyzed with regard to donor or recipient derivation. Laser-assisted microdissection with subsequent short tandem repeat polymerase chain reaction (PCR)-based "genetic fingerprinting" was employed. Combined immunofluorescence and fluorescence in situ hybridization for sex chromosomes was performed for confirmation in cases of gender-mismatched transplantation. Expression of angiogenic factors (FGF-2, PDGF-alpha, PDGF-alpha-receptor, and VEGF-alpha) was analyzed by quantitative real-time reverse-transcription PCR and immunohistochemistry. The inflammatory, nonvascular component of Quilty lesions was completely recipient-derived. Blood vessels were of mixed origin. Different compartments of blood vessels displayed different rates of recipient derivation (endothelium up to 50%, smooth muscle cells up to 15%). Lymphatic vessels were mainly recipient-derived. Of the angiogenic molecules, VEGF-alpha expression was significantly increased in the adjacent myocardium, compared to controls and the Quilty lesions themselves. The inflammatory compartment of Quilty lesions is of recipient origin and shows chimeric neoangiogenesis of blood and lymphatic vessels. VEGF-alpha produced in the adjacent myocardium appears to stimulate the chimeric neoangiogenesis.

Microchip‐Based Cell Lysis and DNA Extraction from Sperm Cells for Application to Forensic Analysis

The current backlog of casework is among the most significant challenges facing crime laboratories at this time. While the development of next-generation microchip-based technology for expedited forensic casework analysis offers one solution to this problem, this will require the adaptation of manual, large-volume, benchtop chemistry to small volume microfluidic devices. Analysis of evidentiary materials from rape kits where semen or sperm cells are commonly found represents a unique set of challenges for on-chip cell lysis and DNA extraction that must be addressed for successful application. The work presented here details the development of a microdevice capable of DNA extraction directly from sperm cells for application to the analysis of sexual assault evidence. A variety of chemical lysing agents are assessed for inclusion in the extraction protocol and a method for DNA purification from sperm cells is described. Suitability of the extracted DNA for short tandem repeat (STR) analysis is assessed and genetic profiles shown. Finally, on-chip cell lysis methods are evaluated, with results from fluorescence visualization of cell rupture and DNA extraction from an integrated cell lysis and purification with subsequent STR amplification presented. A method for on-chip cell lysis and DNA purification is described, with considerations toward inclusion in an integrated microdevice capable of both differential cell sorting and DNA extraction. The results of this work demonstrate the feasibility of incorporating microchip-based cell lysis and DNA extraction into forensic casework analysis.

Whole genome amplification strategy for forensic genetic analysis using single or few cell equivalents of genomic DNA

Evidentiary items sometimes contain an insufficient quantity of DNA for routine forensic genetic analysis. These so-called low copy number DNA samples (<100 pg of genomic DNA) often fall below the sensitivity limitations of routine DNA analysis methods. Theoretically, one way of making such intractable samples amenable to analysis would be to increase the number of starting genomes available for subsequent STR (short tandem repeat) analysis by a whole genome amplification strategy (WGA). Although numerous studies employing WGA have focused primarily on clinical applications, few in-depth studies have been conducted to evaluate the potential usefulness of these methods in forensic casework. After an initial evaluation of existing methods, a modified WGA strategy was developed that appears to have utility for low copy number forensic casework specimens. The method employs a slight, but important, modification of the “improved primer extension preamplification PCR” method (I-PEP-PCR), which we term mIPEP (modified-I-PEP-PCR). Complete autosomal STR and Y-STR (Y chromosome short tandem repeat) profiles were routinely obtained with 5 pg of template DNA, which is equivalent to 1–2 diploid cells. Remarkably, partial Y- and autosomal STR profiles were obtained from mIPEP-treated DNA recovered from bloodstains exposed to the outside environment for 1 year whereas non-mIPEP-treated samples did not produce profiles. STR profiles were obtained from contact DNA from single dermal ridge fingerprints when the DNA was subjected to prior mIPEP amplification but not when the mIPEP step was omitted.

Increased Chimerism of Bronchial and Alveolar Epithelium in Human Lung Allografts Undergoing Chronic Injury

Chimerism on the parenchymal level has been shown for several human allografts, including liver, heart, and kidney, with the integrated recipient-derived cells most likely originating from multipotent bone marrow precursors. We investigated whether chimerism also occurs within epithelial structures of the lung. For this purpose archival tissue biopsies from seven explanted human lung allografts were obtained. We performed laser microdissection of the target structures with subsequent short tandem repeat analysis to detect chimerism within the isolated cells. We found integration of recipient-derived cells in the bronchial epithelium, in type II pneumocytes and in seromucous glands lying adjacent to larger bronchi in all lung allografts studied. Quantitative analysis revealed that the epithelial structures displaying signs of chronic injury, such as squamous metaplasia, showed a markedly higher degree of chimerism (24% versus 9.5%). We therefore conclude that in human lungs, epithelial chimerism occurs at least within bronchi, type II pneumocytes, and seromucous peribronchial glands. Although a bone marrow origin of immigrating host-derived stem cells has been suggested by previous studies in rodents, analysis of lung biopsies from bone marrow-transplanted patients (n = 3) could not prove such delineation in this study. The observation of an enhanced integration of recipient cells into chronically damaged epithelial structures suggests that extrapulmonary precursor cells are able to contribute to pulmonary regeneration.

AmpFℓSTR® Profiler Plus™ and AmpFℓSTR® COfiler™ Analysis of Tissues Stored in GenoFix™, a New Tissue Preservation Solution for Mass Disaster DNA Identification

A preliminary study was conducted to assess the capability of a new alcohol-based tissue fixative, GenoFix, to preserve DNA from biopsy tissues stored at room temperature and/or -20 degrees C in a freezer, for subsequent short tandem repeat (STR) DNA typing analysis. Fresh human smooth muscle samples were stored at room temperature in GenoFix for one month and up to one year and seven months before being processed using the megaplex STR systems, AmpFlSTR Profiler Plus and AmpFlSTR COfiler. Alternatively, muscle tissues in GenoFix were placed at -20 degrees C in a freezer for up to 3 1/2 years following two to three months in the fixative at room temperature. DNA analysis was also carried out on tissues stored in GenoFix for one month at room temperature and subsequently paraffin-embedded and stored at room temperature for four years. The AmpFlSTR Profiler Plus and AmpFlSTR COfiler STR profiles produced, using DNA extracted from all fixed tissue samples, were of very good quality. The fluorescent signals were well balanced across the nine STR loci or six loci comprised in the megaplexes surveyed and profiles showed no differences with those observed for the control blood of the respective donor patients. Continuous exposure to GenoFix at room temperature (up to one year and seven months) did not compromise the STR typing analysis of the fixed tissues. No adverse effects were noted on the STR typeability of tissues fixed with GenoFix and stored at -20 degrees C in a freezer for up to 3 1/2 years. STR profiles generated from the paraffin-embedded tissues fixed in GenoFix were of excellent quality. This preliminary study suggests that GenoFix can be used to store tissue samples at room temperature for up to one year and seven months or at -20 degrees C in a freezer for longer storage (up to 3 1/2 years). This new and odorless tissue fixative promotes tissue and DNA preservation in a very effective manner and as such may prove useful in criminal investigations or mass disaster identifications carried out in remote locations and in which a small or large number of tissue samples are collected for further analyses.