特色酶制剂

分子生物学用

Mesophilic DNA polymerases

Product name

Activity

5′→3′  exonuclease

3′→5′ exonuclease

Optimum temp.

Heat inactivation^a

Strand displacement

Bst DNA Polymerase, Exonuclease Minus

-

-

55-65 °C

80 °C for 20 minutes

++++

NxGen phi29 DNA Polymerase

-

++

30 °C

65 °C for 10 minutes

+++++

Exo-Minus Klenow DNA Polymerase (D355A, E357A)

-

-

37 °C

n.d.

+

^a Indicated treatment results in complete inactivation under standard reaction conditions; n.d., not determined.

Thermophilic DNA polymerases

Product name

Activity

5′→3′  exonuclease

3′→5′ exonuclease

Optimum temp.

Thermostability^a

Fidelity^b

EconoTaq DNA Polymerase

+

-

70-72 °C

n.d.

n.d.

MasterAmp Taq DNA Polymerase

+

-

70-72 °C

10 minutes at 97 °C

0.38-1.82 x10⁴

MasterAmp Tth DNA Polymerase

+

-

68-74 °C

10 minutes at 97 °C

2.2 x 10⁴

LavaLAMP® DNA Enzyme

n.d.

-

68-74 °C

n.d.

n.d.

LavaLAMP® RNA Enzyme

n.d.

-

68-74 °C

n.d.

n.d.

^a Values represent half-lives: 50% of the enzymatic activity is retained after the given time at the stated temperature.

^bDefined as the average number of correct nucleotides a polymerase incorporates before making an error; n.d., not determined.

RNA polymerases

Product name

Activity

5′→3′  exonuclease

3′→5′ exonuclease

Optimum temp.

Heat inactivation

NxGen T7 RNA Polymerase

-

-

37 °C

n.d.

T7 R&DNA Polymerase

-

-

37 °C

n.d.

Poly(A) Polymerase

-

-

37 °C

not recommended

n.d., not determined.

Reverse transcriptases

Enzyme

Activity

Substrates

RNase H activity

Optimum temp.

Heat inactivation

MMLV High Performance Reverse Transcriptase

Synthesises first-strand cDNA

ssRNA, ssDNA

+

37 °C

85 °C for 5 minutes

NxGen M-MuLV Reverse Transcriptase

Synthesises first-strand cDNA

ssRNA, ssDNA

+

37-42 °C

85 °C for 10 minutes

EpiScript RNase H~ Reverse Transcriptase

Synthesises first-strand cDNA

ssRNA, ssDNA

-

37 °C

85 °C for 5 minutes

Enzyme properties

Enzymes for molecular biology

DNA endonucleases

Enzyme

Substrate

Activity

Products

Applications

Optimum temp.

Heat inactivation

Baseline-ZERO DNase

dsDNA and ssDNA

Digests dsDNA or ssDNA to mononucleotides. In presence of Mg²⁺, it cleaves each DNA strand of dsDNA randomly and independently

Mononucleotides

Removing DNA from RNA preparations

37 °C

65 °C for 10 minutes^a

RNase-Free DNase  I

dsDNA and ssDNA

Activated by divalent cations. In presence of Mg²⁺, it cleaves each DNA strand of dsDNA randomly and independently, preferentially adjacent to pyrimidines. In presence of Mn²⁺, it cleaves both strands simultaneously, generating fragments with blunt ends or 1 - to 2-base overhangs.

Oligos and dNMPs with 5′P and 3′OH

•  Removing DNA from RNA preparations •    Random nicking of dsDNA •    DNase footprinting

37 °C

n.d.

^a In the presence of the provided Stop Solution,

n.d., not determined.

DNA exonucleases

Enzyme

Substrate

Activity

Products

Applications

Optimum temp.

Heat inactivation

Exonuclease I (E. coli)

ssDNA

3′→5′ exonuclease that digests ssDNA in the presence of Mg²⁺_

dNMPs

Removal of ssDNA and oligonucleotides.

37 °C

80 °C for 15 minutes

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 RNase H, 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 Nucle-ase or Mung Bean Nuclease to make nested deletions •    Preparation of ssDNA templates for sequencing •    Site-directed mutagenesis •    Preparation of labeled strand-specific probes

37 °C

65 °C for 10 minutes

Exonuclease VII

ssDNA

Exonuclease that digests ssDNA in both 5′→3′ and 3′→ 5′ directions.

dNMPs

Removal of primers and single-stranded oligos.

37 °C

n.d.

Plasmid-Safe ATP-Dependent DNase

linear ssDNA and dsDNA

Selectively digests linear DNA. No activity on nicked or closed-circular dsDNA.

dNMPs

Removal of chromosomal DNA fragments from plasmid, fosmid, and BAC preparations.

37 °C

70 °C for 30 minutes

Rec J Exonuclease

ssDNA

5′→3′ exonuclease that digests ssDNA in the presence of Mg²⁺_

dNMPs

Removal of primers and ssDNA from dsDNA.

37 °C

65 °C for 20 minutes

Nucleases active on both DNA and RNA

Enzyme

Substrate

Activity

Products

Applications

Optimum temp.

Heat inactivation

Terminator 5′-Phosphate-Dependent Exo nuclease

ssDNA or ssRNA

5′→3′ exonuclease that digests ssDNA or ssRNA with 5′-monophosphorylated ends, but not with 5′-OH, 5′-triphosphorylated, or 5′-capped ends

dNMPs or NMPs

•    Removal of 5′-monophosphorylat-ed DNA or primers or oligos •    Enrichment of ssDNA or ssRNA molecules lacking 5′-monophos-phate groups

30 °C (Buffer A) 42 °C (Buffer B)

not recommended

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 preparations •    Removal of host DNA from phage preparations.

25-37 °C

not recommended

RNA nucleases

Enzyme

Substrate

Activity

Products

Applications

Optimum temp.

Heat inactivation

RNase A

ssRNA

Cleaves ssRNA 3′ of pyrimidine residues.

Oligoribonucleo-tides with 3'-cyti-dine or 3'-uridine residues

•    Removal of RNA from DNA preparations •    RNase protection assays •    RNA mapping and structure studies

37 °C (15-70 °C)

not recommended

RNase 1, E. coli

ssRNA

Cleaves ssRNA between all dinucleotide pairs.

NMPs with 5'-OH and 2'，3’-cyclic monophosphate

•    Removal of RNA from DNA preparations •    RNase protection assays •    Mismatch detection of single basepairs in RNA:RNAor RNA:DNA hybrids

37 °C

70 °C for 20 minutes (in presence of 5 mM DTT)

Hybridase Thermostable RNase H

RNA in RNA:DNA hybrid

Cleaves RNA in RNA:DNA hybrid without affecting unhybridised RNA or DNA.

Oligoribonucleo-tides with 5' phosphate and 3' OH.

High-stringency hybrid selection.

45-70 °C

not recommended

RNase R

linear RNA

Digests linear RNA, including the ssRNA end of lariat structures, but not circular RNA or dsRNA with 3′ overhangs <7 nt.

Oligoribonucleo-tides with 5’ phosphate and 3' OH

•Alternative splicing and gene expression studies •    Intron cDNA production •    Intronic screening of cDNA libraries

37 °C

n.d.

n.d., not determined.

Enzyme properties

Enzymes for molecular biology

Ligases

Name of ligase

Cofactor

Ligation template required to ligate

Type of ends ligated

Primary application

Optimum temp.

Heat inactivation

Blunt

Cohesive

NxGen T4 DNA Ligase

ATP

No^a

Yes

Yes

Cloning

4-25 °C

70 °C for 15 minutes

Ampligase Thermostable DNA Ligase

NAD

Yes; DNA only

No

Yes

Template-dependent ligation

45-65 °C

not recommended

Fast-Link DNA Ligase

ATP

No^a

Yes

Yes

Rapid cloning

16-25°C

70 °C for 15 minutes

T4 RNA Ligase 2, Deletion Mutant

Not needed

No

Ligates ss adenylated DNA or RNA to small RNAs

Ligation of RNA to RNA

37 °C

n.d.

CircLigase ssDNA Ligase

ATP

No

Self-ligates (circularises) ssDNA or ssRNA with 5' P and 3' OH

Making ssDNA circles for rolling-circle replication, transcription, and small-RNA sequencing

60 °C

80 °C for 10 minutes

CircLigase II ssDNA Ligase

Not needed

No

Self-ligates (circularises) ssDNA or ssRNA with 5' P and 3' OH

Making ssDNA circles for rolling-circle replication, transcription, and small-RNA sequencing

60 °C

80 °C for 10 minutes

a. These enzymes ligate blunt ends of dsDNA, but ligation is more efficient with cohesive ends,

n.d., not determined.

Phosphatases and kinases

Enzyme

Substrate

Activity

Products

Applications

Optimum temp.

Heat inactivation

RNA5' Polyphosphatase

5'-di or tri-phosphorylated RNA

Removes y and β  phosphates

5'-mono- phosphorylated RNA

•    Ligation-tagging •    Analysis of 5’-end structure

37 °C

n.d.

T4 Polynucleotide Kinase, Cloned

DNA, RNA

Catalyses transfer of Y phosphate of ATP to 5’ terminus of DNA (ds/ss) or RNA (with 3' OH)

Phosphorylated DNA, RNA

Addition of 5' phosphate to DNA or RNA

37 °C

70 °C for 5 minutes

n.d., not determined.

RNA-guided endonucleases

Enzyme

Modifications

Concentration

PAM preference

Type of edit

Guide RNA length

CRISPRcraft S.p. Cas9 Nuclease

One C-terminal NLS, one C-terminal 6 x His tag

10mg/mL(62 µM)

G-rich (NGG)

Blunt double-stranded break

〜97nt

AsCpf1 Nuclease

Two C-terminal NLS, one C-terminal 6 x His tag

10 mg/mL (64 µM)

T-rich (TTTV)

Staggered double-stranded break

〜41 nt

NLS, nuclear localisation signal; PAM, protospacer-adjacent motif.