DNA/cDNA Labeling

Fluorophores and haptens, the latter meaning Biotin, Desthiobiotin, Digoxigenin and Dinitrophenol, are the most commonly used labels for the generation of non-radioactive DNA/cDNA probes. They are enzymatically introduced into DNA/cDNA via modified nucleotides 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).


Label Labeling Kits Single Nucleotides
Fluorophore Fluorescent PCR Labeling Kits
Fluorescent Nick Translation Labeling Kits
Fluorescent Nucleotides
Hapten Biotin & Digoxigenin PCR Labeling Kits
Biotin & Digoxigenin Nick Translation Labeling Kits
Biotin & Digoxigenin 3′-End Oligonucleotide Labeling Kits
Biotinylated Nucleotides Digoxigenin-/DNP-modified Nucleotides
Amine Amine-modified Nucleotides
CLICK CLICK-functionalized Nucleotides
LNA LNA-functionalized Nucleotides → Nucleotides & Nucleosides
Nucleotide dependent
Enzyme Labeling Reagent Kits (w/o labeled nucleotide)  

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

Template Method Labeled Probe Labeling site Enzyme
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
Primer Extension 3’ OH Terminal Deoxynucleotidyl Transferase (TdT)
RNA Reverse Transcription cDNA random Reverse Transcriptase e.g. MMLV

Fluorescent DNA/cDNA Labeling

Table 1: Enzymatic incorporation of fluorescent Nucleotides.

PCR: Polymerase chain reaction with Taq Polymerase; MDA-based WGA: Multiple displacement amplification (MDA)-based whole genome amplification (WGA) with Phi29 DNA Polymerase; NT: Nick Translation with DNAse I / DNA Polymerase I;
Primer Extension: Primer Extension with Klenow 3′-5′exo3′-End Labeling: Incorporation with Terminal deoxynucleotidyl Transferase (TdT); RT: Reverse Transcription with Moloney Murine Leukemia Virus Reverse Transcriptase (MMLV RT)

DNA Labeling cDNA Labeling
Emission color Nucleotide Cat. No. PCR NT Primer Extension 3’ End Labeling RT
Blue AMCA-6-dUTP NU-803-AMCA ✔2 ✔2 n/a n/a n/a
DEAC-dUTP NU-803-DEAC ✔1 n/a n/a n/a
dUTP-ATTO-425 NU-803-425 ✔1 ✔1,2 ✔1 ✔1 n/a
dUTP-XX-ATTO-425 NU-803-XX-425 ✔1 ✔1 n/a n/a n/a
Green dUTP-XX-AF488 NU-803-XX-AF488 ✔1 ✔1 n/a n/a n/a
dUTP-XX-ATTO-488 NU-803-XX-488 ✔1,2 ✔1,2 ✔1,2 ✔1,2 n/a
Fluorescein-12-dUTP NU-803-FAMX ✔1,2 ✔1,2 ✔1,2 ✔1,2 n/a
Rhodamine-12-dUTP NU-803-RHOX ✔1,2 ✔1 ✔1,2 n/a
Yellow-Green dUTP-XX-ATTO-532 NU-803-532 ✔1 ✔1 ✔1 ✔1 n/a
Yellow dUTP-Cy3 NU-803-Cy3 ✔1,2 ✔1,2 ✔1 ✔1 ✔2
dUTP-XX-Cy3 NU-803-XX-CY3 ✔1 ✔