Shop All DNA⁄RNA Modifying Enzymes

Anza™ DNA Blunt End Kit (Invitrogen™)

The Invitrogen™ Anza™ DNA Blunt End Kit is used to convert DNA with cohesive ends to DNA with blunt ends for use in blunt-end ligation reactions. The included Anza™ DNA Blunting Enzyme Mix contains T4 DNA polymerase and Klenow Fragment, while the Anza™ 10X Blunting Buffer contains dNTPs to facilitate the synthesis of blunt ends. Use this kit when the desired vector does not contain a recognition site that would produce blunt ends.

Features:
• Fast 15-minute, room-temperature protocol
• DNA digested with Anza™ restriction enzymes can be used directly in the Anza Blunt End protocol following heat inactivation

The Anza DNA Blunt End Kit is part of the Anza™ Restriction Enzyme Cloning System, unifying the traditional cloning processes. Other Anza DNA Modifying Enzymes include the Anza T4 DNA Ligase Master Mix, Anza T4 PNK Kit, and Anza DNA End Repair Kit.

For more information on the Anza system, visit www.thermofisher.com/anza.

AmpErase™ Uracil N-Glycosylase (UNG) (Applied Biosystems™)

AmpErase® Uracil N-Glycosylase (UNG), part of the GeneAmp® PCR Carry-over Prevention Kit, is a 26 kDa ultrapure, recombinant enzyme encoded by the E. coli uracil N-glycosylase gene, which has been inserted into an E. coli host to direct the expression of the wild type form of the enzyme. The enzyme removes any uracil incorporated into single- or double-stranded DNA.

GeneAmp® PCR Carry-over Prevention Kit
The GeneAmp® PCR Carry-over Prevention Kit (available separately) provides reagents for a simple yet powerful method of ensuring that PCR products cannot be reamplified in subsequent PCR amplifications, thereby preventing false positive results. Features of this kit:

• Enzymatic method stops PCR carryover contamination, which prevents false positive results
• Designed to degrade PCR products from previous PCR amplifications without degrading native nucleic acid templates, which improves amplification
• No interference with any PCR or real-time PCR application
• Optimized for use with GeneAmp® PCR core reagents and GeneAmp® instrument systems

Eliminate PCR carryover contamination
The kit uses an enzymatic reaction analogous to the restriction-modification and excision-repair systems of cells to specifically degrade PCR products from previous PCR amplifications in which dUTP has been incorporated, without degrading native nucleic acid templates. The method used to render PCR products susceptible to degradation involves substituting dUTP for dTTP in the PCR mixture, and pretreating all subsequent PCR mixtures with AmpErase® Uracil N-glycosylase (UNG) prior to PCR amplification. Products from PCR amplification contain uracil, and are readily distinguishable from native thymidine-containing DNA templates. Products from previous PCR amplifications are eliminated by excising uracil residues using UNG, and degrading the resulting abasic polynucleotide with heat. Although UNG is active on single- and double-stranded dU-containing DNA, ribouracil residues in RNA and dUTP itself are not substrates for UNG. AmpliTaq® DNA Polymerase, AmpliTaq Gold® DNA Polymerase, and the other components of the PCR mixture remain intact during UNG treatment, providing PCR amplification free of PCR product carryover. The dU-containing PCR product behaves like dT-containing DNA in blotting, cloning, sequencing, and most other post-PCR analyses.

Anza™ DNA End Repair Kit (Invitrogen™)

The Invitrogen™ Anza™ DNA End Repair Kit is used to convert DNA with cohesive ends to blunt-ended 5’-phosphorylated DNA for use in blunt-end ligation reactions. The included pre-mixed Anza™ DNA End Repair Mix contains T4 DNA polymerase, Klenow Fragment, and T4 PNK (polynucleotide kinase) to minimize pipetting, while the Anza™ 10X End Repair Buffer contains ATP and dNTPs to facilitate the activity of the enzyme mix.

Benefits:
Convenient—pre-mixed enzyme mixture for superior convenience and reduction of pipetting
Efficient—simultaneous blunting and phosphorylation
Compatible—use directly in Anza restriction enzyme digestion reaction mixture following heat inactivation
Fast—end repair complete in 15 minutes

The Anza DNA End Repair Kit is part of the Anza™ Restriction Enzyme Cloning System, unifying the traditional cloning processes. Other Anza DNA Modifying Enzymes include the Anza T4 DNA Ligase Master Mix, Anza T4 PNK Kit, and Anza DNA Blunt End Kit.

TURBO™ DNase (2 U/µL) (Invitrogen™)

TURBO™ DNase cleaves double-stranded DNA nonspecifically to leave 5' phosphorylated oligodeoxynucleotides. It has increased affinity for DNA-binding and remains active in the presence of salt.

Note: this product is just the enzyme. If you would like this enzyme plus reagents to inactivate the enzyme and remove divalent cations post-digestion, please see TURBO DNA-free™ Kit.

Features of TURBO™ DNase include:

• Up to 50x more activity and 350% greater catalytic efficiency
• Efficiently degrades DNA in solutions containing up to 0.25 M salt
• Efficiently digests DNA to oligonucleotides
• Vastly superior in clearing DNA templates from in vitro transcription reactions
• RNase-free and recombinant in origin

Using TURBO™ DNase
DNase I is commonly used to clear DNA contamination from RNA samples prior to RT-PCR. Conventional DNase I has a poor affinity for DNA and cleaves DNA of low concentration very inefficiently. In addition, DNase I is very salt-sensitive; as little as 20 mM NaCl can reduce the activity of the enzyme by 30%. Finally, DNase I is purified from bovine pancreas, one of the richest natural sources of RNase A. The threat of contaminating RNase activity in DNase I preparations requires that the enzyme be exhaustively purified. In spite of these limitations, the DNase I that researchers use today is the very same enzyme that was first characterized by Kunitz more than a half-century ago.

A different DNase with superior properties to wild-type DNase I
TURBO™ DNase was developed using a protein engineering approach that introduced amino acid changes into the DNA binding pocket of wild-type DNase I. These changes markedly increase the affinity of the protein for DNA. The result is a versatile enzyme that has a 6-fold lower Km for DNA, and an ability to maintain at least 50% of peak activity in solutions approaching 200 mM monovalent salt, even when the DNA concentration is in the nanomolar (nM) range. When in vitro transcription reactions are treated with either DNase I or TURBO™ DNase, TURBO™ DNase removes 63x more of the input plasmid DNA template than the wild-type enzyme. The proficiency of TURBO™ DNase in binding very low concentrations of DNA means that the enzyme is particularly effective in removing trace quantities of DNA contamination. This becomes important for complete removal of DNA from a sample, since the cleavable DNA substrate is reduced as the DNase reaction proceeds. TURBO™ DNase thus has a functional advantage over wild-type DNase due to its superior affinity for DNA. This is best exploited in RT-PCR applications, where even a few copies of DNA can lead to a false positive outcome by PCR.

Endonuclease V, T.maritima (5 U/µL) (Thermo Scientific™)

Thermo Scientific Endonuclease V, T. maritima (Endo V) is a 3'-endonuclease involved in DNA repair, which initiates removal of deaminated bases from damaged DNA, including uracil, hypoxanthine, and xanthine.

Endonuclease V is also active toward abasic sites and urea sites, base pair mismatches, flap and pseudo Y structures, and small insertions/deletions in DNA molecules. The cleavage site generated by Endonuclease V is at the second phosphodiester bond 3' to a lesion.

Highlights

• Optimal activity at temperatures of 65 to 70°C

Applications

• High-throughput methods for mutation research
• Studies in mutagenesis and DNA repair
• Mismatch cleavage
• Genotyping

Note

Use of this enzyme in certain applications may be covered by patents and may require a license.

When the enzyme is in excess, the primary nicked products experience a second nicking event on the complementary strand, leading to a double-stranded break. At low concentrations, however, Endonuclease V first nicks a DNA strand at the lesions located closer to the 5'-end of DNA molecule. Single-stranded DNA is cleaved with much lower efficiency. Mg2+ or Mn2+ ions are required for enzyme activity.

T4 Polynucleotide Kinase (10 U/µL) (Thermo Scientific™)

Thermo Scientific™ T4 Polynucleotide Kinase (T4 PNK) catalyzes the transfer of the gamma-phosphate from ATP to the 5'-OH group of single- and double-stranded DNAs and RNAs, oligonucleotides, or nucleoside 3'-monophosphates (forward reaction). The reaction is reversible. In the presence of ADP, T4 Polynucleotide Kinase exhibits 5'-phosphatase activity and catalyzes the exchange of phosphate groups between 5'-P-oligo-polynucleotides and ATP (exchange reaction). The enzyme is also a 3'-phosphatase.

Highlights
• Active in Thermo Scientific restriction enzyme, RT, and T4 DNA ligase buffers

Applications
• Labeling 5' -termini of nucleic acids to be used as:
    --Probes for hybridization
    --Probes for transcript mapping
    --Markers for gel electrophoresis
    --Primers for DNA sequencing
    --Primers for PCR
• 5'-phosphorylation of oligonucleotides, PCR products, other DNA or RNA prior to ligation
• Phosphorylation of PCR primers
• Detection of DNA modification by the [32P]-postlabeling assay
• Removal of 3'-phosphate groups

Notes
• The 5'-termini of nucleic acids can be labeled by either the forward or the exchange reaction.
• Polyethylene glycol (PEG) and spermidine improve the rate and efficiency of the phosphorylation reaction. PEG is used in the exchange reaction mixture.
• Since T4 Polynucleotide Kinase is inhibited by ammonium ions, use sodium acetate to precipitate DNA prior to phosphorylation.

DNase I, Amplification Grade (Invitrogen™)

DNase I, Amplification Grade, digests single- and double-stranded DNA to oligodexyribonuleotides containing a 5' phosphate. DNase I, Amplification Grade, is suitable for eliminating DNA during critical RNA purification procedures such as those prior to RNA-PCR amplification. It is purified and tested for non-detectable levels of RNase contamination. Absence of RNase is tested by performing a ribonuclease assay with RNA ladder.

Application
Removing DNA from RNA and protein preparations.

Specific activity
Specific activity is >10,000 units/mg.

Source
Purified from bovine pancreas

Performance and quality testing
Ribonuclease assay with RNA ladder and ability to digest single-stranded and double-stranded DNA to oligonucleotides are determined.

Unit definition
One unit increases the absorbance of a high molecular weight DNA solution at a rate of 0.001 A260 units/min/mL of reaction mixture at 25°C.

Unit reaction conditions
0.1 M sodium acetate (pH 5.0), 5 mM MgCl2, 50 µg/mL calf thymus DNA, and enzyme in 1 mL for 10 min at 25°C.

Ambion™ RNase A, affinity purified, 1 mg/mL (Invitrogen™)

Ambion® RNase A is an endonuclease that specifically cleaves 3' of U and C residues. Affinity Purified RNase A is intended for critical applications when the absence of DNase and other nonspecific nuclease activities is essential. Supplied in one tube containing 1 mg (1 mg/mL). Note: This preparation contains RNase B, a carbohydrate isoform of RNase A. RNase A is rigorously tested for contaminating nonspecific endonuclease, exonuclease, and protease activity.

HotStart-IT Binding Protein (Thermo Scientific™)

Application
Hot-Start PCR Amplification

HotStart-IT™ Binding Protein is the active component in a novel hot start technology called primer sequestration. In general, hot start PCR methods reduce or eliminate non-specific primer-extension products formed at lower temperatures during assembly of PCR reactions. At these less stringent annealing temperatures, primers may bind non-specifically, which often leads to unwanted amplification products and primer-dimers. In order to resolve this problem, we have produced a high-quality DNA binding protein that is especially useful at sequestering primers at lower temperatures making them unavailable for use by a polymerase (Fig. 1). This primer-sequestration technique effectively blocks DNA synthesis from mis-priming events at lower temperatures. Following the initial denaturation step during PCR, the protein is inactivated and the primers are free to participate in the amplification reaction. Unlike other hot start methods, such as antibodies or chemical modifications, the binding protein is compatible with a variety of thermostable polymerases. HotStart-IT Binding Protein has been designed for PCR applications that demand extremely high specificity and sensitivity and is thoroughly tested for purity and performance.

Properties
HotStart-IT Binding Protein performs well in many standard PCR reaction buffers.

Purity
Free from detectable non-specific nucleases.

Storage Buffer
20 mM Tris-HCl (pH 8.5), 200 mM KCl, 0.1 mM EDTA, 1 mM DTT, and 50% glycerol.

Mass Definition
In general, one microgram of HotStart-IT Binding Protein sequesters about 5 pmol of primers.

Concentration
≥ 10 mg/mL

Source
Recombinant protein expressed in E. coli.

Advantages of HotStart-IT
• Room temperature reaction set-up
• High specificity and sensitivity
• Minimizes amplification of non-specific products and primer-dimers (Fig. 2)
• Ideal for complex templates and multiplex reactions
• Unlike chemically-modified Taq, no extensive heating step is necessary which may damage precious samples
Technology is portable to a polymerase of choice.

Quality Control Polymerase Blocking Assay
This assay compares the amount of Taq DNA Polymerase activity with 2 µg HotStart-IT Binding Protein against its activity with no binding protein. The reaction mixtures contain 0.625 units of polymerase, 1X PCR Reaction Buffer (10 mM Tris-HCl [pH 8.6], 50 mM KCl, 1.5 mM MgCl2), 0.2 mM each dNTP, and 2 pmol of overlapping, extendable oligonucleotides in a 25 µL reaction volume. Following incubation at 25 °C for 4 hours, HotStart-IT Binding Protein blocks at least 90% of the Taq DNA Polymerase activity. (See Fig. 2 for representative data.)

Functional Test
PCR with Taq DNA Polymerase and HotStart-IT Binding Protein shifts production of primer-dimers to a specific target of 306 bp from 1 ng of human genomic DNA relative to Taq DNA Polymerase alone.

Fast DNA End Repair Kit (Thermo Scientific™)

Thermo Scientific Fast DNA End Repair Kit is used for blunting and phosphorylation of DNA ends in just 5 minutes for subsequent use in blunt-end ligation.

All components of the kit contain premixed reagents to reduce pipetting steps and provide convenience. The End Repair Enzyme Mix contains an optimized mixture of T4 DNA Polymerase and Klenow Fragment to achieve highly effective blunting of fragmented DNA, and T4 Polynucleotide Kinase (PNK) for efficient phosphorylation of DNA ends. The 10X End Repair Reaction Mix contains an optimized reaction buffer, ATP, and dNTPs.

Samples such as fragmented genomic DNA (restriction enzyme digested, nebulized or sonicated), restriction enzyme digested plasmid DNA, double stranded cDNA, and PCR products containing dA overhangs are all compatible with the kit.

Features

During the DNA end repair reaction, fragmented DNA is converted into blunt-end DNA containing a 5'-phosphate and 3'-hydroxyl groups. The 5'→3' polymerase activity of the End Repair Enzyme Mix fills-in 5' protruded DNA ends while 3'→5' exonuclease activity removes 3'-overhangs. T4 PNK adds 5'-phosphates to ends of unphosphorylated DNA fragments, such as PCR products. (see Table 1 and Figure 1 below).

Highlights

Efficient - blunting and phosphorylation of 0.5 to 5 µg DNA
Fast—reaction is completed in 5 minutes
Convenient—reaction components are premixed to reduce pipetting steps

Applications

• Blunting and phosphorylation of double-stranded DNA (nebulized DNA, sonicated DNA, restriction enzyme digested DNA, cDNA, PCR products)

Includes

• End Repair Enzyme Mix
• 10X End Repair Reaction Mix

Related Products
CloneJET PCR Cloning Kit

Micrococcal Nuclease (300 U/µL) (Thermo Scientific™)

Thermo Scientific Micrococcal Nuclease (S7 Nuclease) is an endo-exonuclease that digests single-stranded and double-stranded DNA and RNA.
hermo Scientific Micrococcal Nuclease (S7 Nuclease) is a relatively nonspecific endo-exonuclease that digests single-stranded and double-stranded nucleic acids, but is more active on single-stranded substrates. Cleavage of DNA or RNA occurs preferentially at AT or AU-rich regions, yielding mononucleotides and oligonucleotides with terminal 3'-phosphates. The enzyme's activity is strictly dependent on Ca2+.
Applications

• Hydrolysis of nucleic acids in crude cell-free extracts
• Sequencing of RNA (see​ Reference 2)
• Studies of chromatin structure
• A model for protein folding and for structure-function studies

Lambda Exonuclease (10 U/µL) (Thermo Scientific™)

Thermo Scientific Lambda Exonuclease is a highly processive 5'→3' exodeoxyribonuclease. It selectively digests the 5'-phosphorylated strand of double-stranded DNA. The enzyme exhibits low activity on single-stranded DNA and non-phosphorylated DNA, and has no activity at nicks and limited activity at gaps in DNA.

Highlights

• Active in Thermo Scientific PCR buffers

Applications
• Generating single-stranded PCR products for use in: • DNA sequencing
• Analysis of DNA single-strand conformation polymorphism (SSCP)
• Rolling circle amplification

• Producing single-stranded DNA from double-stranded DNA fragments
• Cloning of PCR products

Note

Use of this enzyme in certain applications may be covered by patents and may require a license.

DNase I, RNase-free, HC (50 U/µL) (Thermo Scientific™)

Thermo Scientific DNase I, RNase-free is an endonuclease that digests single- and double-stranded DNA. It hydrolyzes phosphodiester bonds producing mono- and oligodeoxyribonucleotides with 5'-phosphate and 3'-OH groups.

The enzyme activity is strictly dependent on Ca2+ and is activated by Mg2+ or Mn2+ ions.

In the presence of Mg2+, DNase I cleaves each strand of dsDNA independently in a statistically random fashion. In the presence of Mn2+, the enzyme cleaves both DNA strands at approximately the same site, producing DNA fragments with blunt-ends or with overhang termini of only one or two nucleotides.

Highlights

• Recombinant enzyme
• Purified from non-animal host with a lower level of intrinsic RNases

Applications

• Preparation of DNA-free RNA
• Removal of template DNA following in vitro transcription
• Preparation of DNA-free RNA prior to RT-PCR and RT-qPCR
• DNA labeling by nick-translation in conjunction with DNA Polymerase I
• Studies of DNA-protein interactions by DNase I, RNase-free footprinting
• Generation of a library of randomly overlapping DNA inserts. Reaction buffer containing Mn2+ is used

Note

DNase I is sensitive to physical denaturation. Mix gently by inverting the tube. Do not vortex.

Ambion™ RNase H, from E. coli, 10 U/µL (Invitrogen™)

Ambion® RNase H (Ribonuclease H) is isolated from an E. coli strain that over-expresses the gene. RNase H specifically degrades the RNA in RNA:DNA hybrids to produce 3' -hydroxyl and 5' -phosphate terminated products. It is supplied in one tube containing 1,000 U (10 U/ µL). The enzyme will not degrade DNA or unhybridized RNA. RNase H is an integral part of most RNA amplification and NASBA protocols. It can also be used to degrade specific RNAs when the complementary DNA oligo is hybridized, such as poly(A) tail removal from mRNA hybridized to oligo(dT). Ribonuclease H is rigorously tested for contaminating nonspecific endonuclease, exonuclease, RNase, and protease activity.

Unit Definition:
One unit of Ribonuclease H is the amount of enzyme required to increase fluorescence 1.5 RFUs per sec at 37°C using 20 pmol of RNaseAlert® probe coupled to 1,000 pmol of a complementary oligonucleotide as substrate.

RNase T1 (1 U/μL) (Invitrogen™)

Optimized for researchers performing RNA structure, RNA sequencing, protein footprinting, and boundary experiments, Ambion® RNase T1, RNA-Grade cleaves 3' of single-stranded G residues and is tested for purity to ensure that no contaminating ribonuclease activities are present that could cleave at unanticipated sites. Supplied in one tube of 200 µL (1U/ µL). RNase T1 can be used to perform boundary experiments to define the minimal RNA sequence required for selectable activities such as protein binding or catalysis. In addition to applications for RNA structural analysis, RNA-Grade ribonucleases can be used to map protein binding sites on RNAs by comparing cleavage patterns in the presence and absence of an RNA binding protein.

Unit Definitions:
100 units is the amount of enzyme that yields an increase in absorption at 260 nm of 0.01428 units per min at room temperature using 60 µg/mL yeast total RNA as a substrate.

Accessory Products:
Other RNA-Grade ribonuclease available include RNase A (SKU# AM2274), which cleaves 3' of single-stranded C and U residues. Combinations of the single- and double-stranded specific ribonucleases can provide rapid analysis of the physical structure of an interesting RNA.