Molecular Diagnostics: Present and Future
Johan A. Maertens, Kieren A. Marr in Diagnosis of Fungal Infections, 2007
For fungal DNA release and purification, phenol-chloroform extraction procedures were successfully applied (22). However, these protocols were time-consuming and rely on toxic chemicals. Furthermore, the cationic detergent cetyl trimethyl amonium aromide (CTAB) was successfully used to release Aspergillus-ONA (23) from blood and sputum specimens. The use of commercially available kits shortened the duration of DNA extraction (24). Investigators should keep in mind that the sensitivity of these kits varies widely. In our experience, only the Qiamp Tissue Kit® (Qiagen, Hilden, Germany) showed a comparable sensitivity of 10 cells/mL blood to the in-house method, whereas the lowest sensitivity was seen with the DNAzol kit (1000 cells/mL blood). The commercial kits are more expensive than any of the in-house methods. In another study, different DNA extraction methods were comparedfor the extraction of fungal DNA from sera artificially spiked with genomic DNA from Candida species (25). DNA purity and the detection limit were superior for the QIAamp DNA blood kit (Qiagen) and boiling of sera in an alkaline guanidine-phenol-Tris reagent compared to proteinase ढ digestion followed by organic extraction, the HighPure PCR template kit (Roche, Mannheim, Germany) and DNAzol (Sigma, Deissenhofen, Germany).
Organization of the Sites for DNA Attachment to the Nonhistone Proteinaceous Nuclear Skeleton
Isaac Bekhor, Carol J. Mirell, C. C. Liew in Progress in Nonhistone Protein Research, 1985
To isolate proteins remaining bound to DNA and to purify them from other solubilized proteins of the nuclear matrix, the material was ultracentrifuged in a Cs2SO4 density gradient containing the same dissociating agent that had been used for solubilization.65 In these experiments, the matrix was prepared from L cells lysed in the presence of Cu2+ ions (to standardize the isolation procedure). The nuclease treatment was rather mild to obtain relatively long skeleton-associated DNA fragments. This was important for resolution of the DNA-protein band from the band containing free proteins. As shown in Figure 7, protein-label remained associated with DNA fragments solubilized from nuclear matrices by 2% sarcosyl treatment and banded in Cs2SO4 density gradient. This protein-label was found to be resistant to DNAase and RNAase treatments and at least partly sensitive to proteinase treatment followed by phenol-chloroform extraction. Much more protein remained bound to DNA after solubilization with 6 M Gu·HCl or with a mixture containing 2 M urea and 2 M NaCl. As a result, the density of the complexes was shifted toward the lower one comparing to free DNA.
Molecular Methods for the Diagnosis of Fungal Infections
Attila Lorincz in Nucleic Acid Testing for Human Disease, 2016
Although traditional in-house methods have been shown to provide the greatest DNA yields at the lowest cost, these benefits must be balanced by practicality. Methods that may be acceptable for research purposes or for retrospective analyses may be difficult to incorporate into general clinical practice. Therefore, the application of complex enzymatic disruption methods, particularly in conjunction with phenol–chloroform extraction procedures, may not be feasible in a clinical laboratory setting.
A comparison of the genetic and clinical risk factors for arterial hypertension between indigenous and non-indigenous people of the Shoria Mountain Region
Published in Clinical and Experimental Hypertension, 2018
Tatyana Mulerova, Michael Ogarkov, Evgenya Uchasova, Michael Voevoda, Olga Barbarash
Blood for biochemical analysis was taken from the cubital vein in the morning in a fasting state; it was then centrifuged, and the blood serum was frozen and stored at temperatures below freezing. The material was transported to the laboratory in liquid nitrogen-refrigerated containers to avoid thawing. Data related to total cholesterol, high-density lipoprotein cholesterol (HDL-C), triglycerides (TGs), and low-density lipoprotein cholesterol (LDL-C) in serum were evaluated using standard testing systems (Thermo Fisher Scientific Oy, Vantaa, Finland) in a Konelab 30i biochemistry analyzer (Thermo Fisher Scientific). High lipid level was assessed in accordance with the 2012 European guidelines. A plasma fasting glucose level of 5.6–6.9 mmol/L was regarded as a carbohydrate metabolism disorder (fasting hyperglycemia). DNA extraction from blood was performed using a phenol-chloroform extraction technique. Polymorphisms of genes ACE (I/D), ADRB1 (Ser49Gly, A/G, rs1801252), ADRA2B (I/D), MTHFR (C677T, Ala222Val, rs1801133), and eNOS (4b/4a) were assessed by PCR using the following techniques: Snapir et al.; Lima et al.; Salimi et al. (15–17).
Molecular-genetic diagnostics of von Hippel-Lindau syndrome (VHL) in Bulgaria: first complex mutation event in the VHL gene
Published in International Journal of Neuroscience, 2018
Maria Glushkova, Petia Dimova, Iglika Yordanova, Tihomir Todorov, Ivan Tourtourikov, Vanyo Mitev, Albena Todorova
The deletion and duplication screening along the VHL gene was performed by multiplex ligation-dependent probe amplification (MLPA) analysis with standard MLPA kit for the VHL gene (SALSA MLPA P016 VHL probemix), following the manufacturer's instructions [24]. The DNA samples were purified by standard phenol/chloroform extraction. Between 50 and 200 ng of DNAs were diluted in TE buffer up to 4 μl total volume. The diluted samples were subjected to a short denaturation for 10 min in 1 μl denaturing buffer supplied within the MLPA kit. The hybridization with the VHL-specific probes as well as a number of control probes was performed at 60 °C overnight. In the ligation reaction, the hybridized probes were ligated by a specific ligase provided by the manufacturer. The final step represents the PCR amplification of the obtained fragments. The PCR primers, the enzyme dilution buffer and the polymerase are provided in the kit.
Detection and elimination of a novel non-toxigenic Clostridioides difficile strain from the microbiota of a mouse colony
Published in Gut Microbes, 2020
Jeffrey R. Maslanka, Christopher H. Gu, Isma Zarin, Joshua E. Denny, Susan Broadaway, Bryton Fett, Lisa M. Mattei, Seth T. Walk, Michael C. Abt
High molecular weight DNA was extracted from vegetative cells grown overnight in BHIS broth. DNA was isolated by phenol-chloroform extraction as previously described.58 Long-read libraries were prepared by using the SQK-RBK004 version of the Rapid Barcoding Kit (Oxford Nanopore Technologies, Oxford, UK) and sequenced on the Oxford Nanopore MinION using a FLO-MIN106 flow cell. Short-read libraries were prepared using the Nextera DNA Flex Library Prep Kit (Illumina, San Diego, CA) and were sequenced on the HiSeq 2500 using 2 × 125 bp chemistry. The short reads were quality controlled using the Sunbeam59 while the long reads were processed with our in-house pipeline, Nanoflow (https://github.com/zhaoc1/nanoflow). Nanoflow uses the Albacore command line tool to basecall and demultiplex the fast5 files to fastq files. Porechop and Filtlong were used to trim the adapter sequences and subsample to down to 500 Mbps of the highest quality reads (https://github.com/rrwick/Porechop, https://github.com/rrwick/filtlong). Hybrid assembly was performed via Unicycler in Nanoflow. Unicycler uses Spades to generate short read assembly and scaffolds them with a long read assembly generated from Canu and Nanopolish60,61 (https://github.com/jts/nanopolish). CheckM was used to check the quality of draft genomes.62 Phylogenetic analysis of C. difficile strains was generated with an in-house pipeline, coreSNPs (https://github.com/chrgu/coreSNPs). CoreSNPs uses Prokka to annotate the draft genomes and Roary to analyze the pangenome.63,64 Sequences of the core genes were extracted and concatenated to determine the number of single nucleotide variations (SNVs) between genomes via Hamming distances using an in-house R script. Phylogenetic trees and hierarchical clustering trees were generated by FastTree2.65 111 references genomes were gathered from the RefSeq database and 29 clinical isolates from Dr. Fredric Bushman lab’s database.
Related Knowledge Centers
- DNA
- Molecular Biology
- Nucleic Acid
- Polymerase Chain Reaction
- Protein
- Restriction Fragment Length Polymorphism
- Lipid
- Liquid–Liquid Extraction
- Phenol
- Chloroform