Enzyme Properties

Mesophilic DNA Polymerases

Enzyme	5´→3´ Exonuclease	3´→5´ Exonuclease	Nick Translation	Heat Inactivationa	Strand Displacement
RepliPHI™ Phi29 DNA Polymerase	—	++	—	10 min, 65°C	++++
Exo-Minus Klenow DNA Polymerase	—	—	—	20 min, 75°C	+
T4 DNA Polymerase	—	+++	—	20 min, 75°C	—
Terminal deoxynucleotidyl Transferase, Recombinant	—	—	—	20 min, 70°C	—

a. Indicated treatment results in complete inactivation under standard reaction conditions.

Thermophilic DNA Polymerases

Enzyme	5´→3´ Exonuclease	3´→5´ Exonuclease	Thermostabilitya	Fidelityb
MasterAmp™ AmpliTherm™ DNA Polymerase	—	—	NT	NT
MasterAmp™ Taq DNA Polymerase	+	—	9 min, 97.5°C	0.38-1.82 x 104
MasterAmp™ Tfl DNA Polymerase	+	—	40 min, 95°C	8.3-9.0 x 105
MasterAmp™ Tth DNA Polymerase	+	—	20 min, 95°C	2.2 x 104

a. Values represent half-lives: 50% of the enzymatic activity is retained after the given time at the stated temperature.
b.Defined as the average number of correct nucleotides the polymerase incorporates before making an error. NT, not tested.

DNA and RNA Ligases

Enzyme	Ligation Temp	Cofactor	Ligation Template  Required to Ligate	Type of Ends Ligated		Primary Application	Heat Inactivation
				Blunt	Cohesive
T4 DNA Ligase	4°C-25°C	ATP	Noa	Yes	Yes	Cloning	15 min, 65°C
Ampligase™ DNA Ligase	20°C-95°C	NAD	Yes; DNA only	No	Yes	Template-dependent ligation	No; half-life  1 hr, 95°C
CircLigase™ ssDNA Ligase	20°C-65°C	ATP	No	Ligates ssDNA	NAb	Make ssDNA circles	5 min, 100°C
CircLigase™ II ssDNA Ligase	20°C-65°C	Not needed	No	Ligates ssDNA	NAb	Make ssDNA circles	2 min, 100°C
Fast-Link™ DNA Ligase	20°C	ATP	Noa	Yes	Yes	Rapid ligation	10 min, 65°C
Thermostable RNA Ligase	25°C-60°C	ATP	No	Ligates ssRNA	NAb	Ligate ssRNA to ssRNA or ssDNA	5 min, 100°C
T4 RNA Ligase 2, Deletion Mutant	25°C	Not needed	No	Ligates RNA	Ligate DNA or RNA to RNA	cDNA library creation	20 min, 65°C

a. These enzymes ligate blunt ends of dsDNA, but ligation is more efficient on a ligation template, which can be DNA or RNA.
b. Not applicable.

Nucleases Active on Both DNA and RNA

Enzyme	Substrate	Activity	Products	Applications	Heat Inactivation
Terminator™ 5´-Phosphate-Dependent Exonuclease	ssDNA or ssRNA	5´→3´ exonuclease that digests ssDNA or ssRNA with 5´-monophosphorylated ends, but not with 5´-hydroxyl, 5´-triphosphorylated, or 5´-capped ends	dNMPs or NMPs	Removal of 5´-monophosphorylated DNA or primers or oligos. Enrichment of ssDNA or ssRNA molecules lacking 5´-monophosphate groups	10 min, 65°C
OmniCleave™ Endonuclease	ssDNA, dsDNA, or RNA	Endonuclease that efficiently digests DNA and RNA	di-, tri-, and tetra-nucleotides	Removal of DNA and RNA from protein preps. Removal of host DNA from phage preps	No

DNA Endonucleases

Enzyme	Substrate	Activity	Products	Applications	Heat Inactivationa
RNase-Free DNase I (bovine pancreas)	dsDNA and ssDNA	Activated by divalent cations. In presence of Mg2+, it cleaves each DNA strand randomly and independently, preferentially adjacent to pyrimidines. In presence of Mn2+, it cleaves both strands simultaneously, generating fragments with blunt ends of 1-2-base overhangs	Oligos and dNMPs with 5´ phosphate and 3´-OH	Removing DNA from RNA preps. Random nicking of dsDNA. DNase footprinting	20 min, 75°C
Baseline-ZERO™ DNase	dsDNA and ssDNA	Digests dsDNA or ssDNA down to mononucleotides	Mononucleotides	Removing DNA from RNA preps	20 min, 75°C
Endonuclease IV, E. coli	dsDNA with an abasic site	Cleaves sugar-phosphate bond 5´ of an abasic site	dsDNA with single-nucleotide gaps. The cleaved ssDNA strand has a 3´-OH	DNA repair and antitumor drug research. Base Excision Sequence Scanning of DNA containing dUMPs	NT
T4 Endonuclease V	UV-irradiated DNA with thymine dimers	First cleaves N-glycosidic bond 5´ of thymine dimers, then cleaves sugar-phosphate bond 3´ of the abasic site	Nicked dsDNA with an abasic site at the 3´ end of the cut and thymine-dimer bases at the 5´ end of the cut	Research on repair of DNA exposed to UV light	NT
Lambda Terminase	dsDNA with cossites	Cleaves both strands at bacteriophage lambda cos sites	5´ ends with overhangs 12 bases in length	Rapid sizing or restriction mapping of BAC, fosmid, or cosmid clones	NT

DNA Exonucleases

Enzyme	Substrate	Activity	Products	Applications	Heat Inactivationa
Exonuclease I, E. coli	ssDNA	3´→5´ exonuclease	dNMPs	Removal of ssDNA and oligonucleotides.	15 min, 80°C
Exonuclease III, E. coli	dsDNA	3´→5´ exonuclease that digests duplex DNA from the 3´ end of a nick or a blunt or 3´-recessed end; not active on thionucleotides. Exo III also has 3´-DNA phosphatase and apurinic DNA endonuclease activities.	dNMPs and ssDNA on the opposite strand. Partial digestion produces dsDNA having 5´ extensions of ssDNA.	Used with S1 Nuclease or Mung Bean Nuclease to make nested deletions. Preparation of ssDNA templates for sequencing. Site-directed mutagenesis. Preparation of labeled strand-specific probes.	20 min, 70°C
Exonuclease VII	ssDNA	Exonuclease that digests in both 5´→3´ and 3´→5´ directions.	dNMPs	Removal of primers and single-stranded oligos.	10 min, 95°C
Plasmid-Safe™ ATP-Dependent DNase	linear ssDNA and dsDNA	Selectively digests linear DNA. No activity on nicked or closed-circular DNA.	dNMPs	Remove chromosomal DNA fragments from plasmid, fosmid, and BAC preparations.	NT
Lambda Exonuclease	dsDNA	5´→3´ exonuclease that digests dsDNA from 5´-phosphorylated blunt or recessed ends. It has low activity on 5´-hydroxyl ends and is not active on nicked DNA.	dNMPs and ssDNA on the opposite strand. Partial digestion produces dsDNA having 3´ extensions of ssDNA.	Preparation of ssDNA templates for sequencing.	10 min, 75°C
RecBCD Nuclease, E. coli	dsDNA and ssDNA	An ATP- and Mg2+-dependent exonuclease that digests linear DNA in both 5´→3´ and 3´→5´ directions. Not active on nicked or closed-circular dsDNA.	dNMPs	Removal of linear DNA from circular DNA.	NT
Rec J Exonuclease	ssDNA	5´→3´ exonuclease that digests ssDNA with a 5´ phosphate or a 5´OH.	dNMPs	Removal of primers and ssDNA from dsDNA.	20 min, 65°C
T5 Exonuclease	dsDNA and ssDNA	5´→3´ exonuclease that also has single-strand-specific endonuclease activity in presence of 1-10 mM Mg2+ ions. At <1 mM Mg2+, the 5´→3´ exonuclease can digest from a nick in closed-circular dsDNA without digesting the opposite strand.	dNMPs. Circular ssDNA is obtained from closed-circular dsDNA in presence of <1mM Mg2+.	Plasmid mutagenesis. Oligonucleotide site-directed mutagenesis. Removing linear DNA from plasmid preps. Preparation of circular ssDNA	NT

Exonuclease VII	ssDNA	Exonuclease that digests in both 5´→3´ and 3´→5´ directions.	dNMPs	Removal of primers and single-stranded oligos.	10 min, 95°C
Plasmid-Safe™ ATP-Dependent DNase	linear ssDNA and dsDNA	Selectively digests linear DNA. No activity on nicked or closed-circular DNA.	dNMPs	Remove chromosomal DNA fragments from plasmid, fosmid, and BAC preparations.	NT
Lambda Exonuclease	dsDNA	5´→3´ exonuclease that digests dsDNA from 5´-phosphorylated blunt or recessed ends. It has low activity on 5´-hydroxyl ends and is not active on nicked DNA.	dNMPs and ssDNA on the opposite strand. Partial digestion produces dsDNA having 3´ extensions of ssDNA.	Preparation of ssDNA templates for sequencing.	10 min, 75°C
RecBCD Nuclease, E. coli	dsDNA and ssDNA	An ATP- and Mg2+-dependent exonuclease that digests linear DNA in both 5´→3´ and 3´→5´ directions. Not active on nicked or closed-circular dsDNA.	dNMPs	Removal of linear DNA from circular DNA.	NT
Rec J Exonuclease	ssDNA	5´→3´ exonuclease that digests ssDNA with a 5´ phosphate or a 5´OH.	dNMPs	Removal of primers and ssDNA from dsDNA.	20 min, 65°C
T5 Exonuclease	dsDNA and ssDNA	5´→3´ exonuclease that also has single-strand-specific endonuclease activity in presence of 1-10 mM Mg2+ ions. At <1 mM Mg2+, the 5´→3´ exonuclease can digest from a nick in closed-circular dsDNA without digesting the opposite strand.	dNMPs. Circular ssDNA is obtained from closed-circular dsDNA in presence of <1mM Mg2+.	Plasmid mutagenesis. Oligonucleotide site-directed mutagenesis. Removing linear DNA from plasmid preps. Preparation of circular ssDNA	NT

a. NT, not tested.

RNA Endonucleases

Enzyme	Substrate	Activity	Products	Applications	Heat Inactivationa
RNase A	ssRNA	Cleaves ssRNA 3´ of pyrimidine residues.	Oligoribonucleotides with 3´-cytidine or 3´-uridine residues	Removal of RNA from DNA preps. RNase protection assays. RNA mapping and structure studies.	NT
RNase I, E. coli	ssRNA	Cleaves ssRNA between all dinucleotide pairs.	NMPs with 5´-OH and 2´,3´-cyclic monophosphate	Removal of RNA from DNA preps. RNase protection assays. Mismatch detection of single basepairs in RNA:RNA or RNA:DNA hybrids.	15 min, 70°C
RNase III, E. coli	dsRNA	Cleaves dsRNA in presence of Mg2+ to 12-15-bp dsRNA. Cleaves dsRNA in presence of 20mM Co2+ or Mn2+ to 18-25-bp dsRNA.	dsRNA with 5´phosphate and two-base 3´ overhangs with 3´ OH	Random cleavage of long dsRNA to short dsRNA. RNA interference (RNAi). Studies on RNA structure, RNA processing, and maturation.	No
RNase H, E. coli	RNA in RNA:DNA hybrid	Cleaves RNA in RNA:DNA hybrid without affecting unhybridized RNA or DNA.	Oligoribonucleotides with 5´ phosphate and 3´ OH	Elimination of RNA prior to second-strand cDNA synthesis. Removal of poly(A) tails from mRNA hybridized to oligo(DT).	10 min, 65°C
Hybridase™ Thermostable RNase H	RNA in RNA:DNA hybrid	Cleaves RNA in RNA:DNA hybrid without affecting unhybridized RNA or DNA.	Oligoribonucleotides with 5´ phosphate and 3´ OH	High-stringency hybrid selection.	No
RNase T1, Aspergillis oryzae	ssRNA	Cleaves ssRNA 3´ or GMPs	Oligoribonucleotides with 3´-GMP residues	RNA mapping and structure studies. Removal of RNA from DNA preps.	NT

a. NT, not tested.

DNA Glycosylases and Excision Mixes

Enzyme	Substrate	Activity	Products	Applications	Heat Inactivationa
HK™-UNG Thermolabile Uracil-N-Glycosylase	dsDNA or ssDNA containing dUMP	Removes uracil base from dUMP residues in DNA.	Uracil base and abasic DNA. Abasic sites can subsequently be cleaved by AP lyases, such as Endonuclease IV, or by treatment with heat or alkali.	Fragmentation of dUMP-containing DNA. Site-specific cleavage of DNA at dUMP sites. Base Excision Sequence Scanning of DNA containing dUMP residues.	10 min, 65°C
Uracil-DNA Excision Mix	dsDNA or ssDNA containing dUMP	Cleaves sugar-phosphate bond 5´ of dUMPs.	dsDNA with single-nucleotide gaps where dUMP residues have been removed, or ssDNA strands with lengths equal to distances between dUMP residues.	Fragmentation of dUMP-containing DNA. Site-specific cleavage of DNA at dUMP sites. Base Excision Sequence Scanning of DNA containing dUMP residues.	10 min, 65°C