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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