DNA Analysis
Steven H. Y. Wong, Iraving Sunshine in Handbook of Analytical Therapeutic Drug Monitoring and Toxicology, 2017
Sequence information can be obtained by direct sequencing of the DNA locus or more quickly, easily, and inexpensively by probe hybridization. The PCR-based typing technique which is most commonly used at present, is referred to as the reverse dot blot method.49–51 Dot blots involve a series of DNA probes to detect specific target sequences in a given region of DNA. A DNA probe is a small piece of single-stranded DNA (oligonucleotide) which will bind to other single-stranded DNA with the complementary sequence. A sequence-specific oligonucleotide (SSO) probe, also known as an allele-specific oligonucleotide (ASO) probe, is generally composed of 20 to 35 nucleotides. These probes are long enough to confer great specificity, and yet sufficiently short to be destabilized by a single base mismatch, so that it binds only to the exact complement sequence. SSO probes are used to detect the presence or absence of alternative sequence types.
Luminescent Lanthanide Probes as Diagnostic and Therapeutic Tools
Astrid Sigel, Helmut Sigel in Metal Ions in Biological Systems, 2004
Advances in recombinant DNA technology and sensitive methods for analyzing the organization of specific genes are major contributors to genomics. These techniques rely on nucleic acid hybridization probes for the detection of complementary nucleic acid sequences. DNA probes are more and more used in diagnostic medicine, e.g., in the recognition of genetic predisposition to a given disease, virus detection, bacterial identification or antibiotic sensitivity testing. Until the 1990’s, radioisotopes (e.g., 32P, 3H) were common labels for nucleic acid probes, but stability and safety problems led to search for alternative, non-isotopic stains. Truly biochemical methods were proposed, such as biotinylated nucleotide analogues detected by streptavidin or proteins crosslinked to the nucleic acid, but their sensitivity never matched the sensitivity of radio-assays, leaving an opportunity for luminescent assays.
A Survey of Newer Gene Probing Techniques
Victor A. Bernstam in Pocket Guide to GENE LEVEL DIAGNOSTICS in Clinical Practice, 2019
Variability in the estimates of the length of allele fragments (usually within 0.6 to 1.0%) due to the resolving power of agarose electrophoresis used in separating the restricted fragments in single-locus probe testing must be entered into the statistical evaluation of the observed frequency of a restriction fragment in the population. An averaging approach that combines close alleles into the so-called “frequency bins” is used. Advantages of using DNA probes have been clearly demonstrated in a case of disputed paternity when seven probes had been used to produce the cumulative paternity index of 1.4 × 106 that was 316 times higher than that obtainable from the 23 standard blood group markers and HLA.
The current and future applications of in situ hybridization technologies in anatomical pathology
Published in Expert Review of Molecular Diagnostics, 2022
Hoi Yi Leung, Martin Ho Yin Yeung, Wai Tung Leung, King Hin Wong, Wai Yan Tang, William Chi Shing Cho, Heong Ting Wong, Hin Fung Tsang, Yin Kwan Evelyn Wong, Xiao Meng Pei, Hennie Yuk Lin Cheng, Amanda Kit Ching Chan, Sze Chuen Cesar Wong
Lin et al. [67] developed amplification-based single-molecule FISH (asmFISH) which is the combination of a modified smFISH with RCA. First, a pair of DNA probes is used to target the RNA molecule of interest. The probe is then ligated to the RNA by enzymatic reaction, forming a circular DNA for signal amplification via RCA. Lastly, fluorescently labeled probes are hybridized to the amplified DNAs and detected by microscopy. To test the applicability of asmFISH to detect gene expression, HER2 expression in two breast cancer cell lines, MCF-7 and SK-BR-3, were measured. The results matched with the human protein atlas database, therefore, asmFISH could show the difference in levels of gene expression. It also had a higher capacity in discriminating SNPs compared to in situ padlock probe assay. The author demonstrated that asmFISH is applicable in both FFPE and fresh frozen tissue samples, showing the potential in clinical application.
Detection of DNA damage in pigeon erythrocytes using a chromatin dispersion assay
Published in Toxicology Mechanisms and Methods, 2020
Elva I. Cortés-Gutiérrez, Juan A. García-Salas, Martha I. Dávila-Rodríguez, Juan P. Ceyca-Contreras, Michel Cortez-Reyes, José L. Fernández, Jaime Gosálvez
The DNA breakage detection–fluorescence in situ hybridization (DBD-FISH) technique is very sensitive and enables cell-by-cell detection and evaluation of DNA ruptures in whole genome or within precise DNA sequences. After removing the proteins, DNA probes are hybridized and detected. We found earlier that the analysis of erythrocytes from pigeon using DBD-FISH provided a sensitive and reliable test for detecting DNA breaks (Cortés-Gutiérrez et al. 2019). Columba livia has been suggested as a sentinel for assessing the repercussion of air pollutants on urban human populations (Kleinjans and van Schooten 2002; Sicolo et al. 2009). However, microelectrophoretic and FISH methods for assessing DNA damage are time-consuming and require costly and specialized equipment, as well as technical ability (Stricklin et al. 2007; Pereira de Lemos Pinto et al. 2010).
Multifaceted applications of pre-mature chromosome condensation in radiation biodosimetry
Published in International Journal of Radiation Biology, 2020
Usha Yadav, Nagesh Nagabhushana Bhat, Kapil Bansidhar Shirsath, Utkarsha Sagar Mungse, Balvinder Kaur Sapra
For the detection of dicentric chromosomes, G0-PCC spreads from irradiated samples were hybridized with centromere specific Peptide Nucleic acid (PNA) probe. Since the centromeric regions are not clearly visible in prematurely condensed chromosomes, FISH with centromeric probe offered an advantage for visualizing dicentric chromosomes. The chromosomal bodies with one, two or no centromeres were clearly visible as shown in Figure 3(d). Besides, excess fragments are not usually considered as radiation specific compared to dicentrics. Hence demand for radiation specific dicentric signal can be met with G0-PCC using PNA-FISH probes even when conventional DCA is not feasible. These probes are more economical than conventionally available DNA probes. The centromeres staining allow quantification of dicentrics. Dicentric based dose estimations are expected to be more accurate as it is well established that dicentrics are most radiation specific markers with low background frequency and low inter-individual variation. Dicentric is known as gold standard for biodosimetry. Dicentric detection using this method is also important for high doses or high LET exposures wherein a big proportion of cells fail to reach metaphases and thus actual dose estimation can be difficult. There are few reports recently been published on dicentric detection with G0-PCC (Karachristou et al. 2015; M’kacher et al. 2015).
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