Product Class: Other

Mismatch Endonuclease I
neb31 cloned at NEB recombinant 37 70 Heat


Catalog #M0678

Product Introduction



  • DNA Endonuclease
  • Catalyzes the cleavage of some DNA mismatches (T:T, G:G and G:T)
  • Cleaves the 3rd phosphodiester bond on the 5′ side of the mismatched base in both strands leaving a 5 bp overhang with a 3′- OH and 5′- phosphate
  • Best at T mismatches; does not recognize all DNA mismatches   
 

Product Information

Description

Mismatch Endonuclease I is a Mg2+ dependent DNA endonuclease that specifically cleaves mismatched base pairs (T:T, G:G and T:G mismatches). Mismatch Endonuclease I cleaves the 3rd phosphodiester bond on the 5´ side of the mismatched base in both strands, leaving a 5-base pair overhang. While Mismatch Endonuclease I prefers to cleave T:T, G:G and T:G DNA mismatches, it will also readily cleave T:I, G:I and G:U DNA mismatches. Additionally, Mismatch Endonuclease I has been shown to nick the thymine containing strand of T:C DNA mismatches, and cleave T:G and T:U (DNA:RNA) mismatches albeit to a lesser extent than DNA:DNA mismatches.  

Figure 1. Mismatch Endonuclease cleaves T:T, G:T and G:G mismatches in dsDNA. 



(A) Four plasmid constructs (p1-p4) containing a specific mutation at the same locus and differentially phosphorylated primers (either forward or reverse) were used to generate eight double-stranded PCR generated fragments ~672 bp in length (ds1-ds8). Following cleanup (Monarch PCR & DNA Cleanup Kit (NEB #T1030)), the phosphorylated strand of the double-stranded fragments (2.2 µg) was specifically degraded using 5 units of Lambda Exonuclease, incubated at 37°C for 60 minutes, to generate single-stranded oligos of either the top or bottom strand containing either A, T, C or G at the same locus. The single-stranded oligos (ss1-ss8) were then purified using the Oligonucleotide Cleanup Protocol for the Monarch PCR & DNA Cleanup Kit (NEB #T1030).

(B) Purified single-stranded oligos containing either an A, T, C or G can then be mixed and matched to form either perfectly Watson-Crick base-paired DNA (green check marked boxes) or double-stranded DNA oligos containing a single-base mismatch (yellow X boxes). Mismatched dsDNA oligos were generated by mixing the appropriate top and bottom strands (for the mismatch to be created) and re-annealing in 1X NEBuffer 2.1, heated to 95°C, followed by cooling to room temperature, to generate the eight potential DNA mismatches (A:A, A:C, A:G, C:C, C:T, G:G, G:T, T:T). (C) The ability of Mismatch Endonuclease I to cut specific mismatches in dsDNA was queried by incubating 80 units of Mismatch Endonuclease I  with 200 ng of mismatch containing dsDNA, incubated at 37°C for 30 minutes. Products were then run on a 1.2% agarose gel and visualized with ethidium bromide staining. 




Figure 2. Mismatch Endonuclease cleaves T:T, G:T and G:G mismatches in dsDNA. 



The ability of Mismatch Endonuclease I to cut specific mismatches in dsDNA was queried by incubating 80 units of Mismatch Endonuclease I (+M0678), incubated in NEBuffer r2.1 at 37°C for 30 minutes, with double-stranded DNA oligonucleotides containing a single basepair mismatch where the top strand was labeled with a FAM fluorophore (blue traces) and the bottom strand is labeled with a ROX fluorophore (red traces). Samples were analyzed by capillary electrophoresis and cleavage products (+M0678 samples) are evidenced by the shifting of the substrate peaks with regards to the non-enzyme treated samples (-M0678) in the T:T, G:T and G:G mismatched substrates. Moreover, some slight cleavage of the T-containing strand (blue strand) of the T:C mismatch can be seen in 30 minutes. This nicking activity on a T:C mismatch substrate increases over time (up to 18 h, data not shown). 




 

Product Source

An engineered mismatch-specific endonuclease expressed in E. coli.
This product is related to the following categories:

Properties & Usage

Unit Definition

One unit is defined as the amount of enzyme required to cleave 50% of 0.2 pmol of a fluorescently labeled 60mer oligonucleotide duplex containing a single T:T mismatch in 30 minutes at 37°C in a total reaction volume of 20 µl in 1X NEBuffer r2.1.

Reaction Conditions

1X NEBuffer™ r2.1
Incubate at 37°C

NEBuffer™ r2.1
50 mM NaCl
10 mM Tris-HCl
10 mM MgCl2
100 µg/ml Recombinant Albumin
(pH 7.9 @ 25°C)

Storage Buffer

10 mM Tris-HCl
0.1 mM EDTA
1 mM DTT
50% Glycerol
pH 7.4 @ 25°C

Heat Inactivation

70°C for 5 minutes

Features

  • Cleaves T:T, G:G and T:G mismatches in DNA
  • Cleaves T:I, G:I and G:U mismatches in DNA
  • Cleaves T:G and T:U (DNA:RNA) mismatches
  • Nicks thymine strand of T:C mismatches in DNA
 

Product Notes

  1. One unit is defined as the amount of enzyme required to cleave 50% of 0.2 pmol of a single T:T mismatch in 30 minutes at 37°C in a total reaction volume of 20 µl in 1X NEBuffer r2.1. 1 µl of Mismatch Endonclease I contains 80 U, therefore, 1 µl of Mismatch Endonuclease I is able to completely cleave 8 pmol of mismatched DNA.
  2. The addition of molecular crowding agents (PEG, non-specific DNA) and some detergents (Triton X-100) may increase the relative activity of Mismatch Endonuclease I on some mismatches.

Protocols, Manuals & Usage

Protocols

  1. Protocol for Cleavage of Mismatched DNA (T:T, G:G, T:G) (NEB #M0678)*

Tools & Resources

Selection Charts

FAQs & Troubleshooting

FAQs

  1. What is the activity of Mismatch Endonuclease I in other buffers?
  2. Can Mismatch Endonuclease I be inactivated?
  3. What is the activity of Mismatch Endonuclease I at temperatures other than 37°C?
  4. Does Mismatch Endonuclease I require MgCl2?
  5. Can Mismatch Endonuclease I function in the presence of other divalent cations?
  6. Is Mismatch Endonuclease I sensitive to salt?
  7. What mismatched substrates will Mismatch Endonuclease I cleave?
  8. Will Mismatch Endonuclease I cleave consecutive mismatches?
  9. Does Mismatch Endonuclease I cleave mismatches in a DNA/RNA hybrid?
  10. What mismatches will Mismatch Endonuclease I not cleave?
  11. How many picomoles of mismatch DNA can Mismatch Endonuclease I cleave?
  12. How is Mismatch Endonuclease I different than T7 Endonuclease I?
  13. What are the differences between Mismatch Endonuclease I (NEB #M0678) and Authenticase (NEB #M0689)?

Quality, Safety & Legal

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Specifications

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Certificate Of Analysis

The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]

Safety DataSheets

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

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New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.

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