The Precision Medicine Approach in Oncology
David E. Thurston, Ilona Pysz in Chemistry and Pharmacology of Anticancer Drugs, 2021
Measurements of genetic variability can be used to establish a link between the variations and disease, and to predict a patient’s therapeutic response to a drug along with potential toxicological outcomes. After completion of the Human Genome Project (HGP) between 2000 and 2003, the next important step was the discovery of new nonredundant genetic markers known as Single-Nucleotide Polymorphisms or “SNPs”. This involved DNA isolation followed by nucleic acid amplification and product detection. Originally, this was a laborious process involving Southern Blots and manual di-deoxy sequencing. The first technological revolution was based on the polymerase chain reaction (PCR), and included assays such as PCR Restriction Length Fragment Polymorphism (PCR-RLFP), PCR-ligase Detection, PCR Fluorescent Resonance Energy Transfer (PCR-FRET), and the nonamplified FRET assay (Invader Assay). The next improvement resulted from automated sequencing instruments (i.e., DNA Sequencers), which allowed high-throughput nucleotide sequencing and pyrosequencing. Finally, a revolution in SNP analysis resulted from the development of SNP Arrays and accompanying bioinformatics software that enabled large-scale linkages to be identified along with association and copy number studies in a low-cost, high-throughput fashion. Based on these developments, a large range of commercial instruments is now available that allow simultaneous measurement of thousands of SNPs across numerous samples using standard protocols, reagents, and data analysis systems.
Methods in molecular exercise physiology
Adam P. Sharples, James P. Morton, Henning Wackerhage in Molecular Exercise Physiology, 2022
While genomic (gDNA) and mitochondrial DNA (mtDNA) are present in nearly all the body’s cells (an important exception being red blood cells), DNA collection for the purposes of research is typically limited to buccal epithelial cells (cheek cells) or leukocytes (white blood cells). For studies in which blood drawing is a typical procedure, white cells (or the ‘buffy coat’) from a typical non-coagulated blood sample may be used for DNA isolation. If blood sampling is not required for a research study, then collection of buccal cells from the inside of the cheek using a cotton-type swab is a less invasive way to collect DNA. These cells are then processed to burst (‘lyse’) the cells and release the DNA from the cell nuclei, most often using commercially available DNA isolation kits (e.g. DNeasy Blood and Tissue Kit, Qiagen, Manchester, UK), which contain the chemicals needed for cell lysis, protein denaturation (to remove histone and other proteins from the DNA) and purify the remaining DNA. DNA isolation typically requires a couple of hours using the reagents provided in the kits across multiple steps involving centrifugation at high speeds, but many samples can be processed at the same time for efficiency. Once DNA is collected and purified, it can be stored in a sealed tube in a typical refrigerator or freezer for many years; it is one of the most stable biological molecules known. This DNA can then be assessed across a variety of DNA analysis methods typically employed in the field of molecular exercise physiology.
The Genetics of the Frankia-Actinorhizal Symbiosis
Peter M. Gresshoff in Molecular Biology of Symbiotic Nitrogen Fixation, 2018
Early studies on the molecular biology of Frankia were hampered by difficulties with. growing enough cellular material for DNA extraction and also by the low efficiency of classical lysis techniques of Frankia cells. Normand and co-workers37 used a number of different DNA isolation procedures, exploiting such enzymes as lysozyme, protease, neuraminidase, cellulase, helicase, and mutanase, without much effect on lysing a significant portion of cells. Nevertheless, the use of lysozyme in combination with a drastic extraction method (10% SDS at 90°C and vortexing) allowed the extraction of small amounts of DNA. A similar lysis procedure based mainly on the chemical dissolution of the cell wall by hot lauryl sulfate was used by Dobritsa38 and An et al.31 for isolation of Frankia DNA.
The successful strategy of comprehensive pre-implantation genetic testing for beta-thalassaemia–haemoglobin E disease and chromosome balance using karyomapping
Published in Journal of Obstetrics and Gynaecology, 2022
Sirivipa Piyamongkol, Suchada Mongkolchaipak, Pimlak Charoenkwan, Rungthiwa Sirapat, Wanwisa Suriya, Tawiwan Pantasri, Theera Tongsong, Wirawit Piyamongkol
Biopsied cells were washed thoroughly in phosphate-buffered saline (PBS, Cell Signaling Technology, Theera Trading Co. Ltd., Bangkok, Thailand) with 0.1% polyvinyl alcohol (PVA, Sigma-Aldrich, Chiangmai VM Co., Ltd., Chiang Mai, Thailand) before transference to microcentrifuge tubes. DNA extraction was performed using an alkaline lysis buffer protocol (Sermon etal. 1995). 2.5μL of lysis buffer (0.75μL of water, 1.25μL of 0.1M DTT and 0.5μL of 1M NaOH) was added, mixtures were incubated at 60°C for 10min. After that, a neutralisation buffer (2.5μL of 0.4M tricine) was added. Whole genome amplification with multiple displacement amplification (MDA, REPLI-g® Single Cell Kit, Chiangmai VM Co., Ltd., Chiang Mai, Thailand) was then carried out by manufacturer’s instructions. A mixture of 12.5μL of water, 29μL of reaction buffer and 1μL of DNA polymerase (REPLI-g® Single Cell Kit) was added to extracted DNA, making a total volume of 50μL. The mixtures were incubated at 30°C for 2h then at 65°C for 5min to inactivate the reaction.
Molecular diagnosis of toxoplasmosis: recent advances and a look to the future
Published in Expert Review of Anti-infective Therapy, 2021
Marie Gladys Robert, Marie-Pierre Brenier-Pinchart, Cécile Garnaud, Hélène Fricker-Hidalgo, Hervé Pelloux
DNA extraction is the first step in the molecular diagnostic process, and its optimization should not be neglected, otherwise the efficiency of the parasite detection might be reduced. Although manual extraction methods are still widely used in some countries, such as France, the trend is toward automation, which allows this step to be standardized and facilitates its integration into the routine activity of the laboratory [102,113]. In a multicenter evaluation study, Yera et al. compared five commercially available manual and automated T. gondii DNA extraction methods from AF and concluded that if manual and automated methods performed equally at high T. gondii concentrations, results were more contrasted at low concentrations. All automated methods were not equivalent, and the MagNA Pure Compact (Roche) and the BioRobot EZ1 (Qiagen) proved to be more efficient than the manual methods evaluated – i.e. the QIAamp DNA minikit (Qiagen) and the High Pure PCR template preparation kit (Roche) – at low concentrations [114]. Zhao et al. recently compared an automated method – the eMAG (bioMérieux) – with a manual method – the QIAamp DNA Mini Kit (Qiagen) – for artificially spiked CSF, BALF and buffy coat and showed better performance for the automated method, especially for low-concentration samples [115]. Faucher et al. also succeeded in improving the T. gondii DNA extraction of an automated method by integrating a proteinase K pre-treatment step [116]. These data underline the importance of adapting the extraction method to the subsequent PCR method.
Contrasting co-inheritance of alpha and beta mutations in delta beta thalassemia and hereditary persistence of fetal hemoglobin: a study from India
Published in Hematology, 2018
Hareram Pandey, Ravi Ranjan, Kanwaljeet Singh, Amit Sharma, Kamal Kishor, Tulika Seth, Renu Saxena
A total of 52 individuals with raised HbF levels detected during routine screening at Clinical Hematology OPD, All India Institute of Medical sciences(AIIMS), New Delhi in the last 4 years (year 2013–2017) were the study subjects. Patients with XMN1 polymorphism were excluded from our study. Complete history which included the history of jaundice, mass abdomen (left side) and blood transfusion was taken along with written informed consent from the patients. This study was granted ethical approval from AIIMS, Institute ethical committee. About 10 mL of venous blood was drawn in EDTA vial and complete blood count was measured by automated cell analyser. Giemsa-stained peripheral blood smears were examined for red cell morphology. Capillary zone electrophoresis (CZE) (Sebia-Park Technology, Lisses, France) was used for the quantitative assessment of hemoglobin variants HbA, HbF, HbA2. Genomic DNA was isolated from whole blood using Bioserve DNA isolation kit. Asian Indian inversion deletion Gγ(Aγδβ)0-break point A (5′ deletion) and B (3′ deletion) and HPFH-3 were done by GAP-PCR in two tube reaction (one for mutant and one for wild type) using specific primers [9] (Table 1). The primer sequences and PCR conditions for the diagnosis of α-deletion (GAP-PCR) and β-mutation (ARMS-PCR) were selected from the published literature [10–13].
