Epicentre Enzymes

Index

Mesophilic DNA Polymerases

Enzyme 5´→3´ Exonuclease 3´→5´ Exonuclease Nick Translation Heat Inactivationa Strand Displacement
RepliPHI™ Phi29 DNA Polymerase — ++ — 10 min, 65°C ++++
DNA Polymerase I, E. coli + ++ + 20 min, 75°C —
Klenow DNA Polymerase — ++ — 20 min, 75°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.

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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
DisplaceAce™ DNA Polymerase — — 30 min, 65°C NT

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.

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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
E. coli DNA Ligase 5°C-20°C NAD Yes; DNA only Weak activity Yes Make long cDNA 20 min, 65°C
Fast-Link™ DNA Ligase 20°C ATP Noa Yes Yes Rapid ligation 10 min, 65°C
T4 RNA Ligase 37°C ATP No Ligates ssDNA and ssRNA Ligate RNA to DNA Ligate ssRNA to ssRNA or ssDNA 15 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.

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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

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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 cos sites 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
Pvu Rts1I Endonuclease dsDNA Cleaves dsDNA with 5-hydroxymethylcytosines (5-hmc) 3´ ends with overhangs 2 bases in length Analyzing 5-hmc patterns within a genome NT

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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

a. NT, not tested.

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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
RiboShredder™ RNase Blend ssRNA Efficiently degrades all RNA. NMPs Removal or all RNA frm genomic and cloned DNA preps. No

a. NT, not tested.

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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

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