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DNA and cDNA fragments of variable length ("probes") are able to form non-covalent highly specific duplexes with a complementary nucleic acid strand (termed hybridization). When a label is attached to such a hybridization probe it can thus efficiently serve for detection of a defined DNA or RNA target sequence. Labeled DNA/cDNA probes are routinely used e.g. for (fluorescence) in situ hybridization ((F)ISH), micro-array based gene expression profiling or electrophoretic mobility shift assay (EMSA) experiments.

The most commonly used labels for the generation of non-radioactive DNA/cDNA probes are fluorophores and haptens, the latter meaning Biotin, Desthiobiotin, Digoxigenin and Dinitrophenol. Fluorophores are directly detected by fluorescence spectroscopy whereas the visualization of haptens requires secondary reporter molecules (= indirect labels).

Fluorophores and haptens are enzymatically introduced into DNA/cDNA via modified nucleotides (Tab. 1) that are incorporated as substitutes for their natural counterparts in either a one step procedure (introduction of a fluorescent or hapten-modified nucleotide) or a two step procedure (introduction of a reactive group carrying nucleotide and subsequent fluorescent or hapten labeling).

Table 1: Standard enzymatic procedures for the preparation of non-radioactive DNA/cDNA probes.

TemplateMethodLabeled ProbeLabeling siteEnzyme
DNA PCR DNA random Thermophilic polymerase e.g. Taq Polymerase
MDA-based WGA Phi29 DNA Polymerase
Nick Translation DNAse I / DNA Polymerase I
Primer Extension Klenow Fragment 3’-> 5’ exo-, Taq Polymerase
3’ End Labeling 3’ OH Terminal Deoxynucleotidyl Transferase (TdT)
RNA Reverse Transcription cDNA random Reverse Transcriptase e.g. MMLV