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Molecular Approaches Towards the Isolation of Pediatric Cancer Predisposition Genes
Published in John T. Kemshead, Pediatric Tumors: Immunological and Molecular Markers, 2020
This ability to cover large distances in the genome depends on the activity of specific restriction enzymes which cut very infrequently in the human genome. Several such enzymes exist and are listed in Table 1. It will be noted that most of them have, within their recognition sequences, the CpG dinucleotide which, because the human genome is AT-rich, occur five times less frequently than would be expected. 99% of human CpG dinucleotides are methylated, a requirement for the action of the majority of restriction enzymes. The enzymes listed in Table 1, however, require unmethylated DNA as a substrate which only occurs in 1% of the genome. Digestion with these rare cutting enzymes generates DNA fragments which are 0.2 to 2 Mbp long. Conventional agarose gel methods, however, are only capable of separating DNA fragments up to 50 kb long. To cope with the larger fragments generated, pulse field gel electrophoresis (PFGE) has been developed. This work was pioneered by Cantor and colleagues122 working with yeast chromosomes, but the system has since been successfully adapted to separating human DNA. The details of this technique are discussed by van Ommen and Verkerk123 and Anand.124
Acinetobacter — Microbiology
Published in E. Bergogne-Bénézin, M.L. Joly-Guillou, K.J. Towner, Acinetobacter, 2020
Analysis of restriction fragment length polymorphisms by pulsed- field gel electrophoresis (PFGE) is becoming a standard method in hospital epidemiology. In this method, genomic DNA is cut infrequently with enzymes that generate large fragments, which are then separated by special electrophoretic conditions (Birren and Lai, 1993; see Appendix VI). The method has been applied in recent studies of Acinetobacter comparing patterns obtained by restriction enzymes ApaI(Graser et al., 1993; Seifert et al., 1994b, Tankovic et al., 1994), Smal (Allardet-Servent et al., 1989), Apal and Smal (Gouby et al., 1992), Apal and Sfil (L. Dijkshoorn and M. Geneugelijk, unpublished results), and Nhel and Smal (Struelens et al., 1993). Results indicated considerable DNA polymorphism in A. baumannii, even within biotypes, and overall uniformity in outbreaks or multiple isolates from single patients. Longitudinal reproducibility was good (Seifert et al., 1994b) and results were in agreement with those obtained by plasmid typing and PCR fingerprinting (Graser et al., 1993; Struelens et al., 1993; Seifert et al., 1994b). Comparison of PFGE with ribotyping for strains of the A. calcoaceticus-A. baumannii complex showed that PFGE was more discriminatory, but patterns generated by the latter method were not species specific (H. Seifert and P. Gerner-Smidt, unpublished results).
Malassezia
Published in Rossana de Aguiar Cordeiro, Pocket Guide to Mycological Diagnosis, 2019
Reginaldo Gonçalves de Lima-Neto, Danielle Patrícia Cerqueira Macêdo, Ana Maria Rabelo de Carvalho, Carolina Maria da Silva, Rejane Pereira Neves
As previously mentioned, proper identification of Malassezia species can be attained by molecular methods. Many molecular tools have been established for Malassezia identification and also for the detection of intraspecific genetic variation: (a) direct sequencing of rDNA loci (internal transcribed spacers ITS-1 and ITS-2, intergenic spacer IGS-1, large subunit LSU), chitin synthase gene and RNA polymerase subunit 1 gene, (b) PCR-based restriction fragment length polymorphism (PCR-RFLP) of rDNA loci, (c) random amplification of polymorphic DNA (RAPD), (d) denaturing gradient gel electrophoresis (DGGE), and (e) pulsed field gel electrophoresis (PFGE) (Cafarchia et al., 2011). Molecular methods suitable for identification of Malassezia species in clinical mycological laboratories are described in Table 6.3.
CIRCULATING CLONAL COMPLEXES AND SEQUENCE TYPES OF STREPTOCOCCUS PNEUMONIAE SEROTYPE 19A WORLDWIDE: THE IMPORTANCE OF MULTIDRUG RESISTANCE: A SYSTEMATIC LITERATURE REVIEW
Published in Expert Review of Vaccines, 2021
Yara Ruiz García, Javier Nieto Guevara, Patricia Izurieta, Ivo Vojtek, Eduardo Ortega-Barría, Adriana Guzman-Holst
The literature search identified 2,548 potentially relevant records (Figure 2). Following removal of duplicates and screening of titles/abstracts, 98 records were eligible for the detailed screening. Following the full-text screening, 40 records were identified as providing data relevant to the scope of the review (Table 1) [22–61]. Eligible studies reported IPD outcomes in countries where isolates were described by serotype and genotype, in most cases before and after PCVs. Studies used pulsed-field gel electrophoresis or whole-genome sequencing (WGS) combined with multilocus sequence typing (MLST) and electronic Based Upon Related Sequence Types (eBURST) to predict phenotypic traits and describe the clonal structure within serotypes.
Some mutations in the xeroderma pigmentosum D gene may lead to moderate but significant radiosensitivity associated with a delayed radiation-induced ATM nuclear localization
Published in International Journal of Radiation Biology, 2020
Mélanie Ferlazzo, Elise Berthel, Adeline Granzotto, Clément Devic, Laurène Sonzogni, Jean-Thomas Bachelet, Sandrine Pereira, Michel Bourguignon, Alain Sarasin, Mauro Mezzina, Nicolas Foray
Unrepaired chromosome breaks are likely to reflect unrepaired DSB (Joubert et al. 2008). Hence, we examined whether the radiosensitive XPD-mutated fibroblasts elicit DSB repair defect. To this aim, as a first step, we used the pulsed-field gel electrophoresis (PFGE) technique that enables the detection of unrepaired DNA fragments of less than 15 Mbp, independently of any specific DNA repair pathway (Joubert et al. 2008). All the XPD-mutated and control fibroblasts tested showed similar fraction of activity in released DNA fragments (FAR) per Gy assessed immediately after irradiation, suggesting a similar DSB induction rate, consistent with 37 ± 4 DSB per Gy assessed previously in the same irradiation conditions (Foray et al. 1997; Joubert et al. 2008). When expressed as percentage of FAR remaining, the DSB repair rate of the XP16BR, XP17PV and XP26VI fibroblasts was found to be similar (p > .5 for all the combinations). The average FAR remaining after 24 h for these three cell lines was 6.7 ± 3.0%. This value was significantly higher than that of the radioresistant control fibroblasts (0.2 ± 1.0%; p = .03) (Figure 5(A)). The transduction of the XPD gene led to a complete complementation of the DSB repair phenotype in the XPD-transduced counterparts (p > .5 for all the combinations) (Figure 5(A)). Like the radioresistant controls, the XP1BR and XP2NE fibroblasts did not show any significant DSB repair defect when assessed by PFGE (data not shown).
The outcomes of Clostridioides difficile infection in patients with diverticular disease: a nationwide analysis
Published in Scandinavian Journal of Gastroenterology, 2019
Abubaker O. Abdalla, Sai B. Narala, Mohamed A. Abdallah, Rajkumar Doshi, Nageshwara Gullapalli
Clostridioides difficile infection (CDI) is one of the leading causes of hospital-associated infections and has been associated with an estimated 17,000 deaths in 2007 [1]. Previously described risk factors for CDI include advanced age, immunosuppression, renal insufficiency, prolonged hospitalization, antibiotics and proton pump inhibitor use [2–5]. In the United States, CDI caused an estimated half a million infections and approximately 29,000 deaths in 2011. Majority of these cases were health-care-related and more than 80% of deaths related to CDI occurred in patients over 65 years [6]. The estimated total annual CDI-attributable cost in the US is US$6.3 billion. The total annual inpatient stays for hospital management of CDI is 2.4 million days [7]. The emergence of North American pulsed-field gel electrophoresis type 1 strain has resulted in increased morbidity and mortality related to CDI [2].