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
dUTP
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 ✔