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)

Non-radioactive Labeling of DNA and RNA

Labeling of in situ hybridization DNA probes

Hapten-modified Nucleotides for DNA/RNA Labelling

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

Fluorescent Nucleotides

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′exo^–; 3′-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