Practice question

Synthetic oligonucleotides are short single-stranded DNA sequences assembled chemically by solid-phase phosphoramidite chemistry rather than by enzymatic template-directed synthesis. Because they are chemically produced, they emerge unlabeled and require subsequent end-labeling to function as hybridization probes. Common methods include 5' phosphorylation with T4 polynucleotide kinase using [gamma-32P]ATP or 3' tailing with terminal transferase incorporating labeled nucleotides. In contrast, longer probes such as cloned DNA, cDNA, and RNA can be labeled internally during enzymatic synthesis via nick translation, random priming, or in vitro transcription, producing higher specific activities.