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Gene Delivery
Published in Danilo D. Lasic, LIPOSOMES in GENE DELIVERY, 2019
Cationic detergents has been used in DNA purification since the 1950s. Early experiments with cationic liposomes for DNA transfection were less effective than with negative liposomes, and as a result of the toxicity of stearylamine-containing liposomes, these studies were not continued (Fraley and Papahadjopoulos, 1982).
Use of Enzymes in the Downstream Processing of Biopharmaceuticals
Published in Peter Grunwald, Pharmaceutical Biocatalysis, 2019
Another good example of the use of nucleases in the downstream processing of microbial cell-based biopharmaceuticals is seen in the lab-scale isolation of plasmid DNA from the producer E. coli cells. RNase A, in particular, is very popular due to its excellent ability to remove RNA from E. coli cells (Varley et al., 1998; Prazeres et al., 1998). The beneficial impact of RNase clearance in the performance of subsequent chromatographic operations is well known to those who have worked in plasmid DNA purification. However, regulatory concerns with the animal origin of the benchmark RNAse A from bovine pancreas have contributed to the banning of the enzyme from the process scene. An obvious way to circumvent this problem is to use recombinant RNAses, as proposed for example by Voss et al. (2006), who demonstrated the feasibility of using RNase Ba from Bacillus amyloliquefaciens as an alternative to bovine RNase A in the downstream processing of plasmid DNA. Alternatively, many native microbial RNases have been described that could be used for RNA clearance (Hames and Demir, 2015). Surprisingly, neither one of these solutions has been pursued by biopharmaceutical manufacturers.
The relationship polymorphism of gene RFC1 A80G and NSCLP in Sumatera Utara, Indonesia
Published in Cut Adeya Adella, Stem Cell Oncology, 2018
B.Y. Febrianto, U.A. Tarigan, F.B. Buchari, Hidayat
The DNA of the subject was extracted from a blood sample using the Wizard® Genomic DNA purification kit (Promega Corporation, USA) in accordance with the manufacturer’s instructions. Then the DNA was amplified by the PCR method following the procedure used by Lakkakula et al., (2015) with the forward and reverse primer sequences used as follows 5: AGC GGT GA GGT-3 and 5-GGA GGT AGG GGG TGA AG-3. PCR products were cleaved with PCR-RFLP techniques using restriction enzyme HaeII for four hours at 30°C and electrophoresis on agarose gel 3% stained with ethidium bromide.
Liquid biopsy from research to clinical practice: focus on non-small cell lung cancer
Published in Expert Review of Molecular Diagnostics, 2021
Umberto Malapelle, Pasquale Pisapia, Alfredo Addeo, Oscar Arrieta, Beatriz Bellosillo, Andres F. Cardona, Massimo Cristofanilli, Diego De Miguel-Perez, Valeria Denninghoff, Ignacio Durán, Eloísa Jantus-Lewintre, Pier Vitale Nuzzo, Ken O’Byrne, Patrick Pauwels, Edward M. Pickering, Luis E. Raez, Alessandro Russo, Maria José Serrano, David R. Gandara, Giancarlo Troncone, Christian Rolfo
Other of the most popular assays for nucleic acid isolation of EVs is miRNeasy micro kit (Qiagen) that was used for the NGS analysis of EV RNA in serum of early-stage gastric patients and as a result of this study, a signature of 4 miRNAs was found able to improve the diagnostic power of CEA levels [122]. Other technologies such as the MagAttract HMW DNA Kit (Qiagen) have been used for EV DNA purification. Of particular interest, a study performed the analysis of EV DNA and RNA by whole genome, exome, and transcriptome sequencing of EV nucleic acids purified with this kit after ultracentrifugation of pleural effusions in pancreatobiliary cancers. As a result, EV DNA extraction shows high molecular weight double-stranded DNA fragments (>10 kb in size) and alterations in genes such as NOTCH1 and BRCA2 were observed [116]. Moreover, several studies have shown good results with other methodological combinations such as the Total Exosome Isolation kit and Total Exosome RNA and Protein Isolation kit (Life Technologies, Inc., Carlsbad, CA, USA) in plasma and serum [123] or Total Exosome RNA and Protein Isolation kit (Life Technologies, Inc.) after ultracentrifugation methods [124].
Loop-mediated isothermal amplification methods for diagnosis of visceral leishmaniasis (kala-azar) – a systematic review
Published in Expert Review of Molecular Diagnostics, 2020
Gilberto Silva Nunes Bezerra, Walter Lins Barbosa Júnior, Amanda Virgínia Batista Vieira, Amanda Tavares Xavier, Manoel Sebastião Da Costa Lima Júnior, Edeneide Maria Xavier, Elis Dionísio Da Silva, Nilma Cintra Leal, Zulma Maria De Medeiros
The papers were based on a case-control diagnostic study design. They were published between 2009 and 2019. The mixture of geographic locations was not very broad: India (n = 3), Sudan (n = 2), Bangladesh (n = 2), Iran (n = 1), Ethiopia (n = 1), and Brazil (n = 1). There was a variation in the sample size across the included studies ranging from 10 cases/11 controls to 179 cases/88 controls. All studies have primer specificity for the L. donovani complex being kDNA the main target chosen. Regarding the reference test for diagnosing VL, parasitological diagnosis (n = 9) alongside with clinical symptoms (n = 7) were more common. The most applied comparative methods were based on nucleic acid amplification. DNA was extracted from whole blood (n = 8), buffy coat (n = 4), bone marrow aspirate (n = 2), and peripheral blood mononuclear cell (n = 1). In most papers, DNA purification was performed by a commercial kit based in spin-column (n = 9). The results of analytical sensitivity ranged from 1 fg to 10 pg, while the results of clinical sensitivity ranged from 80% to 100% followed by specificity from 95.8% to 100%.
Molecular detection and genotypic characterisation of Streptococcus pneumoniae isolated from children in Malaysia
Published in Pathogens and Global Health, 2020
Shu Ling Goh, Boon Pin Kee, Kartini Abdul Jabar, Kek Heng Chua, Anna Marie Nathan, Jessie Bruyne, Soo Tein Ngoi, Cindy Shuan Ju Teh
Ten strains of S. pneumoniae (three strains with oxacillin zone diameter ≤19 mm and seven randomly selected bacterial isolates which were susceptible to penicillin) were subjected to PCR and DNA sequencing for identification of mutations in pbp1a, pbp2b, and pbp2x genes. Three sets of primers were used to amplify pbp1a (1197 bp), pbp2b (1317bp), and pbp2x (1148 bp) genes as described by Zhou et al. [13]. The 25 µl PCR reaction mixture contained 1X PCR buffer, 1.5 mM of MgCl2, 50 µM of each dNTP, 0.4 µM of each primer, 1.25 U of Taq DNA polymerase (Promega, USA) and 8 µl of DNA template. PCR reaction was programmed to initial denaturation of 94°C for 5 min, 30 cycles of 94°C for 30 sec, 57°C for 30 sec, and 72°C for 1 min, with the final extension of 72°C for 7 min. The PCR products were then purified using MEGAquick-spinTM Total Fragment DNA Purification Kit (Intron Biotechnology) and sequenced by Integrated DNA Technologies, Inc. (Coralville, IA, USA). Sequence results were analyzed by the Basic Local Alignment Search Tool (BLAST) online with R6 as reference strain (GenBank accession no.: NC_003098).