Paul.xu: Created page with "==== Mesophilic DNA Polymerases ==== {| border="1" cellpadding="5" cellspacing="0" |- | Enzyme | 5´→3´ Exonuclease | 3´→5´ Exonuclease | Nick Translation | Heat Inact..."

2018-10-08T15:05:32Z

Created page with "==== Mesophilic DNA Polymerases ==== {| border="1" cellpadding="5" cellspacing="0" |- | Enzyme | 5´→3´ Exonuclease | 3´→5´ Exonuclease | Nick Translation | Heat Inact..."

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==== Mesophilic DNA Polymerases ====

{| border="1" cellpadding="5" cellspacing="0"
|-
| 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 ====

{| border="1" cellpadding="5" cellspacing="0"
|-
| 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 ====

{| border="1" cellpadding="5" cellspacing="0"
|-
| rowspan="2" | Enzyme
| rowspan="2" | Ligation Temp
| rowspan="2" | Cofactor
| rowspan="2" | Ligation Template  Required to Ligate
| colspan="2" | Type of Ends Ligated
| rowspan="2" | Primary Application
| rowspan="2" | 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 ====

{| border="1" cellpadding="5" cellspacing="0"
|-
| 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 ====

{| border="1" cellpadding="5" cellspacing="0"
|-
| 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 ====

==== DNA Exonucleases ====

{| border="1" cellpadding="5" cellspacing="0"
|-
| 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
|}

{| border="1" cellpadding="5" cellspacing="0"
|-
| 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 ====

{| border="1" cellpadding="5" cellspacing="0"
|-
| 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 ====

{| border="1" cellpadding="5" cellspacing="0"
|-
| 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
|}

Enzyme Properties - Revision history

Paul.xu: Created page with "==== Mesophilic DNA Polymerases ==== {| border="1" cellpadding="5" cellspacing="0" |- | Enzyme | 5´→3´ Exonuclease | 3´→5´ Exonuclease | Nick Translation | Heat Inact..."