PubMed · 6337469
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S Athanassiadis, A M Briones. 1983. Evaluation of Tdt methods.. https://doi.org/10.1093/ajcp%2F79.2.272
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A simple enzymatic labeling procedure is described to determine spot quality in oligonucleotide microarrays. By using fluorescently labeled dideoxynucleotides or ribonucleotides as substrate for terminal deoxynucleotidyl transferase (TdT), a single fluorophore can be covalently attached at the 3' end of each oligonucleotide probe molecule in the spot. Fluorescein-12-ddUTP CyTM3-ddUTP Cy5-UTP, and Cy3-UTP were compared as TdT substrates for 3' end labeling an array of 1273 hexamer probes. Cy5-UTP was found to show minimal bias toward probe base composition and is therefore well suited for quantitative analysis of microarray spots where the oligonucleotide probes are coupled via a 5' end linkage to the solid phase.
The nonhomologous end-joining pathway is a major means for repairing double-strand breaks (DSBs) in all mitotic cell types. This repair pathway is also the only efficient means for resolving DSB intermediates in V(D)J recombination, a lymphocyte-specific genome rearrangement required for assembly of antigen receptors. A role for polymerases in end-joining has been well established. They are a major factor in determining the character of repair junctions but, in contrast to 'core' end-joining factors, typically appear to have a subtle impact on the efficiency of end-joining. Recent work implicates several members of the Pol X family in end-joining and suggests surprising complexity in the control of how these different polymerases are employed in this pathway.
An oligonucleotide probe tailed with deoxyadenosine-5'-triphosphate or deoxythymine-5'-triphosphate is detectable with high sensitivity, but has a major drawback--the tail co-hybridizes specifically to complementary sequences. This can be a problem when screening cDNA clones that contain poly(dA) sequences. While it is possible to mask the cDNA tail with unlabeled poly(dA) or poly(A) oligonucleotides, false-positive clones are still produced because complete masking of extremely long (dA) tails is difficult. As a result, only cDNA clones that have extremely long poly(dA) sequences are often obtained by hybridization screening using tailed probes. In this report, we describe an oligonucleotide probe tailed with DIG-labeled nucleotide in combination with deoxyinosine-5'-triphosphate that was highly specific and sensitive to cDNAs. Terminal deoxynucleotidyl transferase efficiently adds dI nucleotides to the 3'-end. The dI of the tails did not pair with any nucleotides under stringent hybridization so that the specificity of hybridization assays remained high without affecting the sensitivity of the test. Colony hybridization experiments demonstrated that there were very few (1 of 80 tested) false positives using this technique. Its use may increase the accuracy of cDNA screening.