Sufficient Amount Of DNA Research Articles

Evaluation of DNA extraction yield from a chlorinated drinking water distribution system.

Desalination technology based on Reverse Osmosis (RO) membrane filtration has been resorted to provide high-quality drinking water. RO produced drinking water is characterized by a low bacterial cell concentration. Monitoring microbial quality and ensuring membrane-treated water safety has taken advantage of the rapid development of DNA-based techniques. However, the DNA extraction process from RO-based drinking water samples needs to be evaluated regarding the biomass amount (filtration volume) and residual disinfectant such as chlorine, as it can affect the DNA yield. We assessed the DNA recovery applied in drinking water microbiome studies as a function of (i) different filtration volumes, (ii) presence and absence of residual chlorine, and (iii) the addition of a known Escherichia coli concentration into the (sterile and non-sterile, chlorinated and dechlorinated) tap water prior filtration, and directly onto the (0.2 μm pore size, 47 mm diameter) mixed ester cellulose membrane filters without and after tap water filtration. Our findings demonstrated that the co-occurrence of residual chlorine and low biomass/cell density water samples (RO-treated water with a total cell concentration ranging between 2.47 × 102–1.5 × 103 cells/mL) failed to provide sufficient DNA quantity (below the threshold concentration required for sequencing-based procedures) irrespective of filtration volumes used (4, 20, 40, 60 L) and even after performing dechlorination. After exposure to tap water containing residual chlorine (0.2 mg/L), we observed a significant reduction of E. coli cell concentration and the degradation of its DNA (DNA yield was below detection limit) at a lower disinfectant level compared to what was previously reported, indicating that free-living bacteria and their DNA present in the drinking water are subject to the same conditions. The membrane spiking experiment confirmed no significant impact from any potential inhibitors (e.g. organic/inorganic components) present in the drinking water matrix on DNA extraction yield. We found that very low DNA content is likely to be the norm in chlorinated drinking water that gives hindsight to its limitation in providing robust results for any downstream molecular analyses for microbiome surveys. We advise that measurement of DNA yield is a necessary first step in chlorinated drinking water distribution systems (DWDSs) before conducting any downstream omics analyses such as amplicon sequencing to avoid inaccurate interpretations of results based on very low DNA content. This study expands a substantial source of bias in using DNA-based methods for low biomass samples typical in chlorinated DWDSs. Suggestions are provided for DNA-based research in drinking water with residual disinfectant.

Perspective: sensitive detection of residual lymphoproliferative disease by NGS and clonal rearrangements—how low can you go?

Malignant lymphoproliferative disorders collectively constitute a large fraction of the hematological cancers, ranging from indolent to highly aggressive neoplasms. Being a diagnostically important hallmark, clonal gene rearrangements of the immunoglobulins enable the detection of residual disease in the clinical course of patients down to a minute fraction of malignant cells. The introduction of next-generation sequencing (NGS) has provided unprecedented assay specificity, with a sensitivity matching that of polymerase chain reaction-based measurable residual disease (MRD) detection down to the 10-6 level. Although reaching 10-6 to 10-7 is theoretically feasible, employing a sufficient amount of DNA and sequencing coverage is placed in the perspective of the practical challenges when relying on clinical samples in contrast to controlled serial dilutions. As we discuss, the randomness of subsampling must be taken into account to accommodate the sensitivity threshold-in terms of both the required number of cells and sequencing coverage. As a substantial part of the reviewed studies do not state the depth of coverage or even amount of DNA in some cases, we call for increased transparency to enable critical assessment of the MRD assays for clinical implementation and feasibility.

One-Pot Synthesis of Defined-Length ssDNA for Multiscaffold DNA Origami.

DNA origami nanostructures generally require a single scaffold strand of specific length, combined with many small staple strands. Ideally, the length of the scaffold strand should be dictated by the size of the designed nanostructure. However, synthesizing arbitrary-length single-stranded DNA in sufficient quantities is difficult. Here, we describe a straightforward and accessible method to produce defined-length ssDNA scaffolds using PCR and subsequent selective enzymatic digestion with T7 exonuclease. This approach produced ssDNA with higher yields than other methods and without the need for purification, which significantly decreased the time from PCR to obtaining pure DNA origami. Furthermore, this enabled us to perform true one-pot synthesis of defined-size DNA origami nanostructures. Additionally, we show that multiple smaller ssDNA scaffolds can efficiently substitute longer scaffolds in the formation of DNA origami.

DONOR HLA GENOTYPING BY NEXT GENERATION SEQUENCING OF DNA FROM URINE-DERIVED DONOR CELLS OF RENAL ALLOGRAFT RECIPIENTS

Introduction: Antibody-mediated rejection (AMR), dominantly induced by anti-HLA antibody, remains the most important cause of long-term graft loss after kidney transplantation. Donor-specific anti-HLA antibody (DSA) can’t often be determined at a specific allele level since historical donor DNA sample is not available and the existing donor HLA typing information is incomplete or even missing. Here, we developed a novel non-invasive technique by selectively expanding donor cells from urine of renal transplant recipients, and thus generating unlimited donor DNA to perform reliable allele-level HLA typing with next generation sequencing method for all HLA loci. Materials and Methods: This study enrolled 28 renal transplant recipients. The fresh midstream urine from each recipient was collected. After urine sample was centrifuged, the supernatant was discarded and washed twice with PBS. The resulting sediment was resuspended in the culture medium. The cells were plated and non-adherent cells were removed. DNA was extracted from the urine-derived donor cells (UDDCs). NGS sequencing is carried out to determine the HLA loci. Results: UDDCs from 26 patients were successfully isolated. High-resolution HLA results were unambiguously determined in 25 out of 26 DNA samples (25/26, 96%) and they are 100% concordant with donor HLA. We showed five HLA sequencing cases of UDDCs (Fig 1). The HLA typing of UDDCs is completely matched with HLA of donor peripheral blood. We further isolated UDDCs from a patient whose donor HLA information is missing at 10 years after kidney transplantation. Based on the UDDCs HLA typing and LSA results, DQA1*05:01 DQB1*02:01 were established as the culprit DSA loci of this patient (Fig 2). Discussion: DNA could be extracted from fresh kidney graft tissues for HLA genotyping; however, this approach is invasive and may aggravate graft injury. The amount of tissues obtained may be minimal and cannot yield sufficient quantity of DNA for HLA typing[1]. Other investigators extract DNA from paraffin embedded tissues or urinary sediments of KTx patients, and mismatched donor antigens could be deduced by subtracting the recipient’s HLA from the allograft HLA[2,3]. However, robust high-resolution HLA typing data can not be obtained by these methods. With an ex vivo expandable source of exfoliated cells, we provide a novel noninvasive urine-based molecular diagnostic method for donor HLA genotyping. Conclusion: The UDDCs-based HLA genotyping strategy successfully establishes complete donor DNA genotypes and provides a feasible and clinically relevant molecular diagnostic method that help to determine DSA loci for kidney transplant patients with historically insufficient donor HLA information.

Using mini-barcodes coupled with high resolution melting (minibar-HRM) method for species discrimination across Pangasianodon gigas, Pangasianodon hypophthalmus and Pangasius larnaudii

Pangasianodon gigas (giant catfish) is well-recognized as a critically endangered species due to overhunting and habitat degradation. Giant catfish aquaculture has expanded to meet the ever-increasing demands with augmented prices. Contamination or substitution of P. gigas products with other related species e.g. Pangasianodon hypophthalmus and Pangasius larnaudii because of their similar morphology but lower cost requires investigation. A method was established to differentiate species by mini-barcodes or microsatellites conjugated with high resolution melting analysis (minibar-HRM or microsatellite-HRM) using mitochondrial genes cytochrome c oxidase subunit I (COI), cytochrome b (Cytb), the nuclear recombination activator gene 1 (Rag1) and two microsatellite loci (Pg-9 and Pg-13). Results revealed that primer sets for the mitochondrial genes PanCytb1 and PanCytb2 showed a significant difference in melting shapes and melting temperature (Tm) between the three species. For nuclear genes, a single locus was not sufficient to discriminate between the three species, and at least two loci such as PanRag1 + Pg9 or PanRag1 + Pg13 were required. The sufficient amount of DNA for species discrimination using minibar-HRM with PanCytb1 and PanCytb2 was suggested as at least 5 pg. This method can also be used for adulteration determination of the P. gigas substitution with P. hypophthalmus and P. larnaudii at greater than 5% adulteration. Findings demonstrated that mini-barcode or microsatellite-HRM offered optimal performance for species discrimination of P. gigas, P. hypophthalmus and P. larnaudii. This approach can be adopted for hybrid detection using PanRag1 + Pg9 or PanRag1 + Pg13-HRM and PanCytb1 or PanCytb2 for matrilineal detection to benefit aquaculture research and quality control on a commercial scale.

Postmortem age estimation via DNA methylation analysis in buccal swabs from corpses in different stages of decomposition\u2014a \u201cproof of principle\u201d study

Age estimation based on the analysis of DNA methylation patterns has become a focus of forensic research within the past few years. However, there is little data available regarding postmortem DNA methylation analysis yet, and literature mainly encompasses analysis of blood from corpses without any signs of decomposition. It is not entirely clear yet which other types of specimen are suitable for postmortem epigenetic age estimation, and if advanced decomposition may affect methylation patterns of CpG sites. In living persons, buccal swabs are an easily accessible source of DNA for epigenetic age estimation. In this work, the applicability of this approach (buccal swabs as source of DNA) under different postmortem conditions was tested. Methylation levels of PDE4C were investigated in buccal swab samples collected from 73 corpses (0–90 years old; mean: 51.2) in different stages of decomposition. Moreover, buccal swab samples from 142 living individuals (0–89 years old; mean 41.2) were analysed. As expected, methylation levels exhibited a high correlation with age in living individuals (training set: r2 = 0.87, validation set: r2 = 0.85). This was also the case in postmortem samples (r2 = 0.90), independent of the state of decomposition. Only in advanced putrified cases with extremely low DNA amounts, epigenetic age estimation was not possible. In conclusion, buccal swabs are a suitable and easy to collect source for DNA methylation analysis as long as sufficient amounts of DNA are present.

High-throughput Preparation of DNA, RNA, and Protein from Cryopreserved Human iPSCs for Multi-omics Analysis.

We describe the procedure to isolate genomic DNA, RNA, and protein directly from cryopreserved induced pluripotent stem cell (iPSC) vials using commercially available solid-phase extraction kits, and we report the relationship between macromolecule yields and experimental and storage factors. Sufficient quantities of DNA, RNA, and protein are recoverable from as low as 1 million cryopreserved cells across 728 distinct iPSC lines suitable for whole-genome sequencing, RNA sequencing, and mass spectrometry experiments. Nucleic acids extracted from iPSC stocks cryopreserved up to 4 years maintain sufficient quantity and integrity for downstream analysis with minimal genomic DNA fragmentation. An expected positive correlation exists between cell count and DNA or RNA yield, with comparable yields recovered between cells across different cryostorage timespans. This article provides an effective way to simultaneously isolate iPSC biomolecules for multi-omics investigations. © 2020 Wiley Periodicals LLC. Basic Protocol 1: QIAshredder and AllPrep DNA/RNA/protein mini kit extraction and subsequent DNA quantification and quality analysis Basic Protocol 2: Broad-range RNA quantification and quality assay using QuBit 4 Fluorometer and associated kits.

Spindle cell/sclerosing rhabdomyosarcoma: a clinicopathological study of 20 cases

Objective: To study the clinicopathological features and immunophenotype of spindle cell/sclerosing rhabdomyosarcoma (SRMS) in adults and children, as well as its correlation with the expression and gene-mutations of MYOD1. Methods: Twenty cases of SRMS were collected at Xijing Hospital, Fourth Military Medical University from 2009 to 2019. These cases were evaluated for clinical, pathological, and immunohistochemical features. MYOD1 gene sequencing was performed on 12 cases with available tissue and sufficient DNA quantity using Sanger sequencing. Results: The 20 patients included 12 children and 8 adults, 11 males and 9 females, with an age range of 8 months to 85 years (mean 22 years). Most of them presented with a painless, progressively enlarged solid mass. The tumors occurred in head and neck (7 cases), abdominal and pelvic cavity (7 cases, including 4 in abdominal cavity, 2 in pelvic cavity, 1 in abdominal and left thoracic cavity), upper limb (5 cases, including 2 in left shoulder, 1 in right armpit, 1 in right humerus, and 1 in left forearm), and the back (1 case). The diameter of these tumors ranged from 2.5 to 20 cm, with a mean of 6.2 cm. Histologically, all of the tumors were mainly composed of spindle cells arranged in fascicles, and in 7 cases, at least in part, arranged in herringbone pattern, resembling adult fibrosarcoma. Foci reminiscent of interstitial sclerosing were presented in 4 cases, pseudovascular structures in 2 cases, loosely myxoid stroma in 4 cases, and varying degree of necrosis in 9 cases. A various number of spindled or polygonal rhabdomyoblasts were observed between spindle cells in 3 cases.Among them,16 cases showed spindle cell morphology, 2 cases showed scleroisng morphology, and 2 cases showed a hybrid phenotype of spindle, sclerosing and primitive undifferentiated areas. Immunohistochemically, the tumor cells were positive for desmin, Myogenin and/or MyoD1, but negative for CKpan, ALK1, CD34, EMA, HMB45, SMA, H-cald and S-100. Four cases (4/12) harbored a homozygous or heterozygous MYOD1 (p.L122R) mutation. MYOD1-mutant SRMS usually had diffuse and strong nuclear MyoD1 positivity. Follow-up was available in 12 cases, ranged from 1 to 51 months. At the end of follow-up period, 3 patients died of the disease, 3 patients developed local recurrences, 2 patients survived with disease. Conclusions: SRMS is a rare type of rhabdomyosarcoma, and more commonly occurs in the head and neck of children than adults. MYOD1-mutant SRMS usually had diffuse and strong nuclear MyoD1 positivity, frequently associated with a more aggressive behavior.

Improved Protocol for DNA Extraction from Subsoils Using Phosphate Lysis Buffer.

As our understanding of soil biology deepens, there is a growing demand for investigations addressing microbial processes in the earth beneath the topsoil layer, called subsoil. High clay content in subsoils often hinders the recovery of sufficient quantities of DNA as clay particles bind nucleic acids. Here, an efficient and reproducible DNA extraction method for 200 mg dried soil based on sodium dodecyl sulfate (SDS) lysis in the presence of phosphate buffer has been developed. The extraction protocol was optimized by quantifying bacterial 16S and fungal 18S rRNA genes amplified from extracts obtained by different combinations of lysis methods and phosphate buffer washes. The combination of one minute of bead beating, followed by ten min incubation at 65°C in the presence of 1 M phosphate buffer with 0.5% SDS, was found to produce the best results. The optimized protocol was compared with a commonly used cetyltrimethylammonium bromide (CTAB) method, using Phaeozem soil collected from 60 cm depth at a conventional agricultural field and validated on five subsoils. The reproducibility and robustness of the protocol was corroborated by an interlaboratory comparison. The DNA extraction protocol offers a reproducible and cost-effective tool for DNA-based studies of subsoil biology.

Improving sustainable use of genetic resources in biodiversity archives.

Tissue sample databases housed in biodiversity archives represent a vast trove of genetic resources, and these tissues are often destructively subsampled and provided to researchers for DNA extractions and subsequent sequencing. While obtaining a sufficient quantity of DNA for downstream applications is vital for these researchers, it is also important to preserve tissue resources for future use given that the original material is destructively and consumptively sampled with each use. It is therefore necessary to develop standardized tissue subsampling and loaning procedures to ensure that tissues are being used efficiently. In this study, we specifically focus on the efficiency of DNA extraction methods by using anuran liver and muscle tissues maintained at a biodiversity archive. We conducted a series of experiments to test whether current practices involving coarse visual assessments of tissue size are effective, how tissue mass correlates with DNA yield and concentration, and whether the amount of DNA recovered is correlated with sample age. We found that tissue samples between 2 and 8 mg resulted in the most efficient extractions, with tissues at the lower end of this range providing more DNA per unit mass and tissues at the higher end of this range providing more total DNA. Additionally, we found no correlation between tissue age and DNA yield. Because we find that even very small tissue subsamples tend to yield far more DNA than is required by researchers for modern sequencing applications (including whole genome shotgun sequencing), we recommend that biodiversity archives consider dramatically improving sustainable use of their archived material by providing researchers with set quantities of extracted DNA rather than with the subsampled tissues themselves.

Validation of presumptive tests for non-human blood using Kastle Meyer and Hemastix reagents

Kastle Meyer and Hemastix reagents are presumptive tests commonly used in forensic casework for the detection of blood, and their suitability has been reviewed in numerous publications. However, studies to date have focused on the validation of these tests on human blood alone, and no published work has looked at the sensitivity, specificity and effect on DNA analysis when using these reagents to presumptively test for animal blood. The aim of this study was to validate the two reagents for use with animal blood, and compare their performance in order to choose the best test based on the circumstances in wildlife crime investigation.The sensitivity, specificity, stability and robustness of the methods were assessed by experiments with dilutions of animal blood (from 1:4 to 1:65536) using direct and indirect (rub) tests, potential interfering substances, blood sources from different species and aged blood. The effects of the two reagents on subsequent DNA analysis were also investigated.During the direct tests, Kastle Meyer showed a higher sensitivity, detecting blood down to a dilution of 1:16,384, one order of magnitude lower than Hemastix. However during the rub test, Hemastix showed a higher sensitivity, detecting blood down to a dilution of 1:64 on porous materials while Kastle Meyer was positive only down to a dilution of 1:16. Moreover, when using the same swab for presumptive testing and DNA extraction, Hemastix testing allowed amplification of a sufficient amount of DNA for species identification at its limit of sensitivity on porous materials (1:64) while Kastle Meyer inhibited most amplification of DNA at its less sensitive limit of 1:16 dilution. On the other hand, Hemastix showed a much lower specificity, producing false positive results when exposed to tomato, potato, rust, avian uric acid, bleach and sink rot, while Kastle Meyer only produced a faint positive reaction from potato. Both tests performed equally well detecting fresh blood of different animal species. The stability test gave comparable results among the tests except for aged fish blood stains, where the Kastle Meyer test performed poorly.Owing to its ease of use, higher sensitivity, and lack of interference with downstream DNA analysis, and despite its reduced specificity compared to Kastle Meyer, the Hemastix method is more appropriate for use in wildlife crime investigations. Positive results would always be confirmed with DNA analysis and the low interference of the reagent will allow the use of a single swab for presumptive testing and DNA sampling.

The importance of forensic storage support: DNA quality from 11-year-old saliva on FTA cards.

Storage conditions influence the integrity of the recoverable DNA from forensic evidence in terms of yield and quality. FTA cards are widely used in the forensic practice as their chemically treated matrix provides protection from the moment of collection to the point of analysis with current STR typing technology. In this study, we assess the recoverability and the integrity of DNA from 11-year-old saliva on FTA cards using a forensic quantitative real-time polymerase chain reaction (qPCR) commercial assay. The quality after long-term storage was investigated in order to evaluate if the FTA device could assure enough stability over time, applying some internally validated quality criteria of the STR profile. Furthermore, we used a 3D interpolation model to combine the quantitative and qualitative data from qPCR to calculate the minimum optimal DNA input (MODI) to add to the downstream PCR reaction based on the quantitative and qualitative data of a sample. According to our results, when saliva sample is properly transferred onto FTA cards and then correctly stored according to the manufacturer's instructions, it is possible to recover sufficient amounts of DNA for human identification even after more than a decade of storage at ambient temperature. Degradation affected the quality of results especially when the Degradation Index exceeds the value of 2.12, requiring modifications of the standard internal workflow to improve the genotyping quality. Above this value, the application of a "corrective factor" to the PCR normalization process was necessary in order to adjust the recommended manufacturer's PCR DNA input taking into account the degradation level. Our results demonstrated the importance to consider in predictive terms the parameters obtained with the real-time quantification assay, both in terms of quantity (DNA concentration) and of quality (DI, inhibition). Informatics predictive tools including qPCR data together with the variables of storage duration and conditions should be developed in order to optimize the DNA analysis process.

Touch DNA: impact of handling time on touch deposit and evaluation of different recovery techniques: An experimental study

“Touch DNA” is DNA obtained from biological material transferred from a donor to an object or a person during physical contact. This particular kind of evidence could play an essential role in forensic laboratory work and is considered an important tool for investigators. Even though the principal aspects of “Touch DNA” have been extensively studied, to date, there are few reports in the research field of DNA retrieval from garments that have been worn. This study aimed to investigate the “handling time”, analyzing particularly the minimum contact time required to deposit a sufficient amount of DNA on a garment to produce an interpretable profile of the “handler”. Moreover, three different sampling techniques were compared (“dry swab”, “cutting out”, and “adhesive tape”) with the aim of defining the technique that guarantees the best recovery of the three methods tested. Analyzing the data of this experimental model, a “handling time” of two seconds is enough to release sufficient DNA on to a garment to obtain a complete profile. Moreover, this study demonstrated that when targeting for foreign DNA, the sample area should be narrowed down as much as possible to the smallest area possible to maximize target DNA recovery.

Whole genome analysis of the common pathogens associated with microbial keratitis

Background Microbial Keratitis is a serious infection of the cornea and a major cause of corneal opacity and vision loss worldwide. Whole-genome sequencing (WGS) of keratitis bacterial isolates has the ability to provide a wealth of clinically-valuable information. The potential of metagenomics has also been evaluated as current techniques for pathogen identification are laborious, time-consuming and associated with poor culture rates, making metagenomics an exciting future prospect. Methods/Results Samples from keratitis patients were collected and isolates grown in agar before DNA was extracted from pure-cultures and WGS data was obtained from a total of 30 isolates, including common causative agents Pseudomonas aeruginosa and Staphylococcus aureus. Libraries were prepared using a miniaturised Nextera XT protocol and sequencing was carried out on Illumina’s NextSeq platform. Genomic analysis was then based around MLST-typing, comparative genomics, and the identification of key genes inferring antibiotic resistance and virulence. Our pathogens exhibited diverse evolutionary lineages and a range of virulence-associated genes, providing insight into pathogenicity. The ability to obtain sufficient DNA quantities for metagenomics sequencing was also assessed using various host depletion and microbiome enrichment techniques. Discussion/Conclusion The clinical role of next-generation sequencing is currently limited due to associated costs and required computational resources, however in the future this method could replace current techniques for pathogen identification. WGS provides important insights into the genomes of the common keratitis infectious agents, improving understanding of the aetiology of corneal infections and metagenomics shows future potential to be the centre of culture-independent pathogen identification.

Non-destructive DNA extraction from aphids: the application in virus - vector studies of Banana bunchy top virus (BBTV)

Banana bunchy top virus (BBTV), one of the most devastating viruses of banana, is transmitted by the banana aphid, Pentalonia nigronervosa. The high degree of morphological similarity between P. nigronervosa and the related experimental vector P. caladii makes it difficult to distinguish the two species. DNA barcoding can be used as an alternative for the rapid identification of these aphid species. However, standard molecular identification techniques for small insects usually require maceration of the sample to obtain sufficient amounts of DNA, resulting in the loss of voucher specimens. In this study, a non-destructive DNA extraction method for aphids was optimised, allowing the specimens to remain intact for use as vouchers for further morphological studies. Sufficient DNA concentrations were obtained from the aphid extractions and were used for species identification through direct sequencing of PCR products. Extracted DNA was further used to detect BBTV in the insect vector, P. nigronervosa. Approximately 78% of the aphids collected from symptomatic plants in infected plantations in KwaZulu-Natal tested positive for BBTV, as well as some aphids collected in other African countries. The BBTV viral strain identified from South Africa grouped with the “South Pacific” clade. This non-destructive DNA extraction method can be used in an early detection management strategy, especially in epidemiology and virus-vector studies of Banana bunchy top disease (BBTD).

DNA Extraction and Polymerase Chain Reaction.

DNA extraction and polymerase chain reaction (PCR) are the basic techniques employed in the molecular laboratory. This short overview covers various physical and chemical methods used for DNA extraction so as to obtain a good-quality DNA in sufficient quantity. DNA can be amplified with the help of PCR. The basic principle and different variants of PCR are discussed.

Survey of local fauna from endemic areas of Northern Queensland, Australia for the presence of Mycobacterium ulcerans.

Buruli ulcer (BU), regionally known as the Daintree ulcer or Bairnsdale ulcer is caused by the environmental pathogen Mycobacterium ulcerans (MU). This disease is characterized by extensive and painless necrosis of skin and soft tissue with the formation of large ulcers and has been reported in >33 countries worldwide. This organism is geographically restricted and in Australia, the disease has been reported primarily in coastal Victoria and the Mossman-Daintree areas of northern Queensland. Australia is the only country where nonhuman cases of BU have been confirmed. The common ringtail possums and mountain brushtail possums have been suggested as potential animal reservoirs of MU in coastal Victoria, Australia. The exact mode of transmission of this disease remains unknown. In this study, we surveyed local fauna from endemic areas of northern Queensland, Australia, for the presence of MU in scat samples. We collected 140 bandicoot, four white-tailed rats, and two possum scat samples from 56 overnight trapping sessions. Samples were examined for the presence of MU DNA by the polymerase chain reaction. Two out of five samples did not contain a sufficient amount of DNA to detect IS2606 and the ketoreductase B (KR) domain of the mycolactone polyketide synthase gene, which is represented by higher cycle threshold (Ct) values for IS2404 shown in table below. Despite of having desired Ct values for IS2404, one IS2404 positive sample possibly contained DNA of closely related M. ulcerans subspecies with lower copy number of IS2606 that do not commonly cause disease in human. All three targets: IS2404, IS2606 and KR were detected from the remaining two scat samples. We confirm the presence of M. ulcerans DNA in the scat samples collected from a Buruli ulcer endemic region of Northern Queensland, Australia.

Isolation of High-Molecular-Weight DNA from Mouse Yolk Sacs and the Like.

Often, genotyping of mouse embryos is required, and a small part, such as the yolk sac, can be used for this purpose. Here, DNA samples are prepared from extra-embryonic tissues by digestion with Proteinase K and subsequent extraction. The yolk sac of mid-gestation or later-stage embryos provides a sufficient amount of DNA for Southern analysis. Small tissues of a few hundred cells are used for genotyping early postimplantation- and preimplantation-stage embryos by polymerase chain reaction (PCR).

DNA barcodes from snake venom: a broadly applicable method for extraction of DNA from snake venoms.

DNA barcoding is a simple technique used to develop a large-scale system of classification that is broadly applicable across a wide variety of taxa. DNA-based analysis of snake venoms can provide a system of classification independent of currently accepted taxonomic relationships by generating DNA barcodes specific to each venom sample. DNA purification from dried snake venoms has previously required large amounts of starting material, has resulted in low yields and inconsistent amplification, and was possible with front-fanged snakes only. Here, we present a modified DNA extraction protocol applied to venoms of both front- and rear-fanged snakes that requires significantly less starting material (1mg) and yields sufficient amounts of DNA for successful PCR amplification of regions commonly used for DNA barcoding. [Formula: see text].

Analysis of the embryonic media drop as a non-invasive atlernative to blastocyst biopsy in preimplantation genetic testing for aneuploidy

Current methods for preimplantation genetic testing of aneuploidy (PGT-A) require the biopsy of cells from the developing embryo. Improvements in clinical outcomes with PGT-A have been reported worldwide. Nevertheless, biopsy procedures are invasive with the potential to impact embryonic developmental competence, are costly to the patient and time consuming for the embryology laboratory. The aim of this study was to blindly analyze the embryonic media drop as a non-invasive alternative to blastocyst biopsy for PGT-A. Prospective cohort blinded study. Infertility patients planning in vitro fertilization (IVF) with intracytoplasmic sperm injection (ICSI) and PGT-A between January-March 2018 (n=14) were consented with IRB approval to media drop analysis following blastocyst development. Blastocysts were biopsied on either day 5 or day 6 of development upon identification of an inner cell mass. Media drops (20μl) were collected at two different developmental timepoints, from day 3 to day 5 of embryo culture, as well as day 5 to day 6 of blastocyst culture. Trophectoderm (TE) biopsies and media drops were collected separately and blindly analyzed for chromosome constitution using the next generation sequencing, VeriSeq™ platform (Illumina). Chromosome numeration from these two embryonic DNA sources were decoded and compared to evaluate the clinical efficiency of media drop analysis for PGT-A. DNA amplification was observed in 100% of the media drop samples (n=91), with 80 (87.9%) resulting in sufficient quantities of DNA for a reliable PGT-A result. Concordance with TE biopsy was confirmed in 72 (90%) of the media drop samples for embryo sex and 64 (80%) of the media drop samples for the overall PGT-A result. The 10% sex discordance represented euploid males diagnosed as euploid females due to the presence of maternal DNA contamination in the media drop from fallen cumulus cells following zona pellucida expansion. Interestingly, this sex concordance was only observed in day 3 to day 5 media drop samples, and not in any of the day 5 to day 6 media drop samples. The remaining 10% discordance represented media drop samples that were misrepresented with a mixture of different autosomal aneuploidy, including a few media drop samples with the reverse aneuploidy, which would be the expected result following mitotic cell division errors. Our clinical data suggests that embryonic DNA is present and can be utilized for PGT-A in media drops at later stages of blastocyst development. Overall, the accuracy and reliability of blastocyst DNA in media drops was observed to be lower than intact nucleated TE biopsied cells. However, ongoing improvements in both the embryology and genetic laboratories are focused on closing this gap in order to offer infertility patients an option of non-invasive PGT-A.