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Comparative Genomic Hybridization and Copy Number Abnormalities in Breast Cancer
Published in Brian Leyland-Jones, Pharmacogenetics of Breast Cancer, 2020
The first CGH experiments employed metaphase chromosomes as the representation onto which information was mapped (28,33). While useful, the genomic resolution was limited by the nonlinear organization of DNA along metaphase chromosomes. Thus, chromosome CGH has now been largely supplanted by array CGH, in which information is mapped onto arrays of DNA probes (Fig.1). The initial array CGH platforms comprised large genomic cloned probes, such as yeast artificial chromosomes (YACs) (34), bacterial artificial chromosomes (BACs), and PI-derived artificial chromosomes (PACs) (29), or shorter cDNA sequences spotted on glass slides (35). More recently, synthetic oligonucleotide probes have been used successfully (36–39). Several platforms are summarized in Table 1 and in the following sections.
Enteric Pathogens
Published in Victor A. Bernstam, Pocket Guide to GENE LEVEL DIAGNOSTICS in Clinical Practice, 2019
Drawbacks of oligonucleotide probes include an overnight incubation for maximum sensitivitylaborious isolation of viral nucleic acids
In situ Hybridization Histochemistry
Published in Edythe D. London, Imaging Drug Action in the Brain, 2017
Martin K.-H. Schofer, James P. Herman, Stanley J. Watson
Several characteristics of oligonucleotide probes render them quite attractive for in situ experiments. First, they are by far the easiest of three types to acquire, as they can be generated to any published nucleotide sequence. Most molecular biology departments have an automatic oligonucleotide synthesizer available; in addition, many commercial sources offer custom oligonucleotide synthesis. Second, oligonucleotides are quite easy to use; generally, in situ hybridization using oligonucleotides does not require extensive molecular biological skill or experience. Third, probe composition and length can easily be designed on paper by the investigator, allowing for analysis of specific regions of the mRNA in question. However, it is required that probe design be very carefully planned to ensure specificity for the particular RNA of interest (Agarwal et al., 1981). For example, one does not want to use oligonucleotides directed against sequences common to many DNA or RNA molecules, such as DNA binding domains of steroid receptors and related compounds (Evans, 1988) or highly conserved transmembrane spanning domains of membrane-bound receptors (Bunzow et al., 1988).
Mutation patterns of epidermal growth factor receptor gene in non-small cell lung cancer among Egyptian patients
Published in Egyptian Journal of Basic and Applied Sciences, 2022
Wafaa H. Elmetnawy, Mona Qenawi, Salwa Sabet, Heba Bassiony
Extracted DNA was amplified by Multiplex PCR amplification of specific DNA sequences using biotinylated primers. Then, the amplification products were analyzed by gel electrophoresis. The amplified products were hybridized to a test strip containing allele-specific oligonucleotide probes immobilized as an array of parallel lines (Precise selective hybridization of specific sequences onto Strip Assay). Malignant tumor and control samples were tested for specific EGFR mutations using ASO strip assay that targets three mutations affecting codon 719 in exon 18 (G719A, G719C, and G719S), grouped deletions within exon 19, insertions within exon 20, and the individual mutations T790M, L858R, L861Q, and S768I. The 30 investigated mutations in the four exons of EGFR gene are listed in Table 1.
Cytogenetic and molecular genetic methods for chromosomal translocations detection with reference to the KMT2A/MLL gene
Published in Critical Reviews in Clinical Laboratory Sciences, 2021
Nikolai Lomov, Elena Zerkalenkova, Svetlana Lebedeva, Vladimir Viushkov, Mikhail A. Rubtsov
The studied DNA is fragmented, either physically or enzymatically, and the obtained fragments are ligated to NGS adapters for sequencing (Figure 6(b)). Targeted sequencing can be performed, such as whole-exome sequencing (WES), or frequently rearranged genes can be sequenced using special panels [128]. Both WGS and targeted sequencing are used to detect translocations, and the choice of approach depends on the tasks and resources available to the laboratory. The hybridization of biotinylated oligonucleotide probes to genes of interest can predominantly be used for enrichment. To date, a KMT2A-targeted panel has been developed to study all variants of its rearrangements [8]. This panel consists of 2688 overlapping oligonucleotide probes (capture probes) that cover the entire KMT2A sequence. The possibility of performing targeted sequencing using such a panel is much wider than with LDI-PCR because the detection of translocation does not depend on the presence of restriction sites in the partner genes and is not very sensitive to the fragmentation of the original DNA. Using this panel, a minor breakpoint cluster in KMT2A rearrangements was identified [8].
A profile on cobas® EGFR Mutation Test v2 as companion diagnostic for first-line treatment of patients with non-small cell lung cancer
Published in Expert Review of Molecular Diagnostics, 2020
Susana Torres, Álvaro González, Alberto Jacobo Cunquero Tomas, Silvia Calabuig Fariñas, Macarena Ferrero, Danielle Mirda, Rafael Sirera, Eloisa Jantus-Lewintre, Carlos Camps
In order to select each targeted mutation, the test uses oligonucleotide probes which are labeled with a fluorescent dye (reporter), together with a quencher molecule that absorbs fluorescent emissions from intact probe. During each cycle of amplification, the complementary probe binds to the single-stranded DNA sequence in the amplicon and is consequently cleaved by the nuclease activity of the DNA polymerase; this cleavage separates the reporter dye from the quencher, allowing fluorescence emission of a characteristic wavelength by the reporter. Despite the fact that three different reporter dyes are used to label the mutations targeted by the test, amplification of the seven targeted EGFR sequences are detected independently.