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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
Nick translation and random primer extension are the most commonly used methods for labeling cDNA probes. Nick translation involves incubation of double-stranded probe molecule (separated from any bacterial sequences by restriction digestion-electrophoresis) with DNase I and Eschericha coli DNA polymerase I. The DNase creates nicks (base excisions) in the cDNA, which is both extended on the 5′ side by the exonuclease activity and filled in on the 3′ side by the polymerase activity of the DNA polymerase I. Radioactive nucleotides included in the translation mix are incorporated during the filling-in process. Random priming, on the other hand, takes advantage of the ability of oligonucleotides to prime DNA synthesis on single-stranded templates. S ingle-stranded cDNAs (again free of bacterial sequences) are incubated with random single-stranded DNA fragments 6 to 12 nucleotides in length in the presence of the Klenow fragment of DNA polymerase I and labeled and unlabeled nucleotides. Generally, random priming introduces a larger proportion of radioactive nucleotides into probes than does nick translation, resulting in two to tenfold higher specific activities (Sambrook et al., 1989).
The Use of Molecular Hybridization Techniques as Tools to Evaluate Hepatic Fibrogenesis
Published in Marcos Rojkind, Connective Tissue in Health and Disease, 2017
Mark Α. Zern, Mark J. Czaja, Francis R. Weiner
If a radiolabeled cDNA (complementary DNA) probe is prepared from a purified unique mRNA species, hybridization of this cDNA to a total RNA fraction quantitates sequences in the total RNA that are in turn complementary to the DNA. When this recombinant, double-stranded cDNA is to be utilized as a probe, it must be labeled with a radioactive compound. One such reaction is termed nick-translation,23 in which labeled dXTPs are substituted enzymatically for nucleotides in the DNA by bacterial DNA polymerase I. This method produces DNA probes with high radioactive activity (more than 108 cpm/μg DNA). Such probes can detect less than 1 pg of a specific nucleic acid sequence (DNA or RNA). Thus, molecular hybridization can be performed with [32P]-labeled probes under conditions which are 1000 times more sensitive on a weight basis than standard radioimmunoassays for the detection of specific proteins.
Characterization of the 2 M NaCl-Resistant Chromatin Fraction from Chicken Erythroid Cells
Published in Isaac Bekhor, Carol J. Mirell, C. C. Liew, Progress in Nonhistone Protein Research, 1985
Peter C. Hentzen, Isaae Bekhor
Nick translation of purified DNA (DNA-P and DNA-T) was done according to Norman and Bekhor.53 The specific activity of nick-translated DNA using (3 H)-dCTP (24 Ci/mmol, New England Nuclear) ranged from 1 to 2 × 106 cpm/μg DNA.
DBD-FISH, an effective marker for detecting genotoxicity in buccal mucosa exfoliated cells of patients with oral cancer
Published in Toxicology Mechanisms and Methods, 2021
Elva I Cortés-Gutiérrez, Jorge G. Garza Molina, Martha I Dávila-Rodríguez, Pablo Zapata Benavides, José M Faz Eguía, Ricardo M Cerda-Flores
To estimate total DNA damage, a whole-genome DNA probe was originated from buccal epithelial cell pellets using a DNA isolation kit for mammalian blood (Roche Diagnostics Corporation, Indianapolis, IN, USA). An aliquot (1-µg) of the DNA sample was labeled with biotin-14-2′-deoxyUridine 5′-TriPhosphate (dUTP) utilizing a commercial nick-translation kit (Roche Diagnostics Corporation, Indianapolis, IN, USA). The hybridized DNA probe was identified by incubation for 30 min with streptavidin-Cy3 streptavidin-Cy3 (Sigma Chemical Co., Darmstadt, Germany). Hybridizations and posthybridization washes were performed according to the manufacturer’s specifications. Finally, the slides were counterstained with 1 μg/ml 4′,6-DiAmidino-2-PhenylIndole (DAPI) in Vectashield mounting medium (Vector Laboratories, Burlingame, CA, USA). Cells also were exposed to 100 μM hydrogen peroxide (H2O2) at room temperature for 10 min to induce DNA single-strand breaks (ssbDNA) (positive control).
Clinical genomic profiling to identify actionable alterations for very early relapsed triple-negative breast cancer patients in the Chinese population
Published in Annals of Medicine, 2021
Liye Wang, Qinglian Zhai, Qianyi Lu, Kaping Lee, Qiufan Zheng, Ruoxi Hong, Shusen Wang
Rearrangements of ROS1 (6q22) and EPHA7 (6q16) were independently detected using a laboratory-developed dual-colour break-apart probe (BAP) strategy probe set. 5′ and 3′ of probes of ROS1 and EPHA7 were labelled with red and green fluorescence bacterial artificial chromosome (BAC), respectively. BAC clone probes flanking the target genes were obtained from Invitrogen (Waltham, MA). DNA from each BAC probe was labelled with fluorochromes by nick translation. FFPE sections were deparaffinized, pre-treated and then hybridized with the denatured probes. Following overnight incubation, the slides were rinsed, stained with 4′,6-diamidino-2-phenylindole (DAPI), mounted and analysed using a Nikon fluorescence microscope (Nikon ECLIPSE 80i, Tokyo, Japan).
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
There are a variety of probes available for ISH. Complementary DNA (cDNA) and RNA probes (riboprobes) are made from cloning using a cloned nucleic acid sequence inserted into a plasmid vector. It is simpler to label by nick translation for cDNA probes but they must be denatured before applying to the tissues. Riboprobe is a product from the probe cDNA template by ribonucleic acid polymerase. Oligonucleotide probes are synthetic oligonucleotide DNA probes with the length of 20 to 50 bases [16].