Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I¶
Why this mattered¶
Nick translation labeling made recombinant DNA experimentally legible. Rigby, Dieckmann, Rhodes, and Berg showed that E. coli DNA polymerase I could use its coupled exonuclease and polymerase activities to replace nucleotides along nicked DNA with α-^32P-labeled precursors, producing probes with very high specific activity from both circular and linear DNA. The shift was not conceptual in the sense of discovering hybridization, but practical and enabling: cloned DNA fragments could now be converted into sensitive, sequence-specific reagents in vitro, reproducibly and at scale.
That capability transformed methods such as Southern blotting, plaque and colony screening, restriction mapping, and later Northern blots and in situ hybridization. Before such probes were routine, finding one sequence within a complex genome or a recombinant library was slow and often indirect; afterward, a cloned fragment could be used to detect its complement in genomic DNA, RNA, cells, or libraries. This helped turn molecular biology from bulk biochemistry into sequence-directed analysis.
Its importance is best seen in the breakthroughs it made ordinary: isolation of genes from genomic and cDNA libraries, mapping of gene copy number and rearrangements, detection of viral and oncogene sequences, RFLP analysis, and the probe-based logic that underlay positional cloning and early genome mapping. Later nonradioactive labels, random priming, PCR probes, and sequencing-based assays displaced nick translation in many workflows, but they inherited its central paradigm: labeled nucleic acids as precise molecular addresses for finding, measuring, and comparing genetic information.
Abstract¶
(no abstract available)
Related¶
- cite → Detection of specific sequences among DNA fragments separated by gel electrophoresis — Nick translation labeling supplies high-specific-activity DNA probes for Southern's gel-blot detection of specific DNA sequences.