In situ Hybridization Histochemistry
Edythe D. London in Imaging Drug Action in the Brain, 2017
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
Marcos Rojkind in Connective Tissue in Health and Disease, 2017
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
Isaac Bekhor, Carol J. Mirell, C. C. Liew in Progress in Nonhistone Protein Research, 1985
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).
Comprehensive characterization of a transgene insertion in a highly repetitive, centromeric region of Anopheles mosquitoes
Published in Pathogens and Global Health, 2023
Matteo Vitale, Chiara Leo, Thomas Courty, Nace Kranjc, John B. Connolly, Giulia Morselli, Christopher Bamikole, Roya Elaine Haghighat-Khah, Federica Bernardini, Silke Fuchs
Ovaries at Christopher stage III from 18/33-h half-gravid females were dissected from 7 wild-type and 8 Ag(PMB1) individuals and fixed for 24-h at Room Temperature (RT) in Carnoy’s solution. Polytene chromosome preparations were obtained according to Xia et al. [23]. Chromosome spread quality check was performed using a phase-contrast microscope and then immersed in Liquid Nitrogen. Subsequently, they were dehydrated in a series of ethanol (50%, 70%, 90%, 100%) for 5 minutes each and then allowed to air dry at RT and stored at – 20°C. To perform Fluorescent In Situ Hybridization (FISH) on each chromosome preparation, 800 ng of pBac [3xP3-DsRed]b2eGFP::I-PpoI124L plasmid DNA [11] was labeled with dUTP-Cy5 (GE Healthcare) by nick translation reaction (ROCHE). The reaction mixture was incubated at 15°C for 90 minutes and stopped by heating at 65°C for 10 minutes. PCR reaction (F-TATCGGCTGCAACATCAAAC, R-ACAGAGGTTGTTGAGGAACCA) was used to generate probes from the gene AGAP004670 [24]. DNA probes were precipitated by adding 1/10 volume of 3 M NaAC and 2.5 volume of 100% ethanol and centrifuged at 14,000 g for 20 minutes at 4°C. The Probe was then resuspended in hybridization buffer that was pre-warmed at 37°C. FISH was performed as previously described by Xia et al. [23].
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).
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