Product Class: Other

Authenticase®
NEBU 42 No


Catalog #M0689

Product Introduction

Graphic of mechanism

Mixture of structure-specific nucleases capable of recognizing and cleaving outside mismatches and indels

  • Recognizes indels (insertions and/or deletions) as well as single base mismatches: C/C, T/C, A/C, T/G, G/G, T/T and A/A
  • Applications:
    • Error-correction in oligonucleotide synthesis
    • Mismatch Detection Assay
  • Learn more about how Authenticase can enhance error correction during gene synthesis in our NEB Inspired blog post.

Product Information

Description

Authenticase is a proprietary mixture of structure-specific nucleases capable of recognizing and cleaving outside mismatch and indel (insertion and/or deletion) regions, ranging from 1-10 basepairs (bp) on double-stranded DNA. The formulation has limited non-specific activity on homoduplex regions of DNA.  Authenticase can be used as an error-correction reagent in oligo-based PCR gene assembly by enzymatically removing “mistakes” prior to the final renaturation and amplification step (i.e. removes mismatch/indel errors caused by oligonucleotide synthesis). Alternatively, Authenticase can replace T7 Endonuclease I in the mismatch detection assay used to assess the efficiency of genome editing.

FIGURE 1: Mechanism of Authenticase

Image of Mechanism

Authenticase cleaves dsDNA carrying mismatches or indels, leaving either 3′ or 5′ overhangs.




FIGURE 2: Applications of Authenticase

Images of application use

Authenticase is a mixture of structure-specific nucleases capable of recognizing and cleaving outside mismatch and indel (insertion and/or deletion) regions, ranging from 1–10 basepairs (bp) on double-stranded DNA. The formulation has limited non-specific activity on homoduplex regions of DNA. Authenticase can be used as an error-correction reagent in oligo-based PCR gene assembly by enzymatically removing “mistakes” prior to the final renaturation and amplification step. Alternatively, Authenticase can replace T7 Endonuclease I in the mismatch detection assay used to assess the efficiency of genome editing (S1 is the starting material.  P1 and P2 are products of Authenticase digestion.).




FIGURE 3: Authenticase Reduces Error Rate as compared to CorrectASE™

Image of error rates

Sixteen oligonucleotides (ranging in size from 50–60 nucleotides) were synthesized by IDT® Inc. and used to amplify an MBD gene (645 bp). PCR amplicons were processed further to reduce the error frequency in fragments, as suggested in the manual. Error-corrected fragments were cloned into vectors using NEBuilder® HiFi DNA Assembly Cloning Kit (NEB #E5520) and colonies were isolated from selective plates (LB +amp). 12 DNA plasmids were purified from each set of experiments (12 E. coli colonies per set: 1) No enzyme treatment; 2) Treatment with Authenticase and 3) treatment with CorrectASE™) followed by Sanger DNA sequencing. Authenticase reduces the error rate from 1 out of 645 bases to 1 out of 1935 bases as compared to CorrectASE which was 1 out of 1,419 bases.




FIGURE 4: Detection of Mutations by Authenticase versus T7 Endonuclease I

Images of Detection

Mismatch detection comparisons were performed using Authenticase or T7 Endonuclease I. Pools of heteroduplex DNA with a variety of mismatches or indels (e.g. A/C mismatch, T/T mismatch, 2-bp indel, etc.) were artificially created by mixing sequence-defined PCR amplicons followed by heating and re-annealing. Thus, heteroduplex substrates with pre-set mutation frequencies (around 21–25% mutant and 75–79% WT) were established for each variant tested. Mismatch cleavage by Authenticase or T7 Endo I was performed and the reaction products were resolved on an Agilent® Bioanalyzer®. The estimated mutation frequency (% cleavage) was calculated based on the molarity of the observed cleavage products and starting substrate and then graphed with the expected ratio shown as a solid black reference line. Authenticase performs as well or better than T7 Endonuclease I. Authenticase outperforms T7 Endonuclease I if the types of mutations in the population are single base or 2-bp mutation . Authenticase performs as well as T7 Endonuclease I if the types of mutations in the population are indels (insertions/deletions) or more than 3-bp mutations.

This product is related to the following categories:
DNA Repair Enzymes and Structure-specific Endonucleases Products
This product can be used in the following applications:
Genome Editing Applications

Reagents Supplied

Reagents Supplied

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration
  • M0689S     -20    
      Authenticase® M0689SVIAL -20 1 x 0.025 ml 25 reactions
      Authenticase DNA Annealing Buffer B2831SVIAL -20 1 x 0.7 ml 5 X
      Authenticase Reaction Buffer B2832SVIAL -20 1 x 0.5 ml 10 X
  • M0689L     -20    
      Authenticase® M0689LVIAL -20 1 x 0.125 ml 125 reactions
      Authenticase DNA Annealing Buffer B2831SVIAL -20 1 x 0.7 ml 5 X
      Authenticase Reaction Buffer B2832SVIAL -20 1 x 0.5 ml 10 X

Properties & Usage

Reaction Conditions

1X Authenticase Reaction Buffer
Incubate at 42°C

1X Authenticase Reaction Buffer
10 mM Tris-HCl
10 mM MgCl2
100 µg/ml Recombinant Albumin
(pH 8 @ 25°C)

Storage Buffer

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

Heat Inactivation

No

Features

  • Reduces number of colonies that need to be screened and saves time
  • Can replace T7 Endonuclease I in mismatch detection assay

Protocols, Manuals & Usage

Protocols

  1. Error Correction During Gene Synthesis (NEB #M0689)
  2. Mismatch Detection Assay (NEB #M0689)
  3. Supplemental Protocol 1: Generation of DNA fragments by PCR assembly of pooled oligos (NEB #M0689)
  4. Supplemental Protocol 2: Using colony PCR to identify positive clones (NEB #M0689)

Manuals

The Product Manual includes details for how to use the product, as well as details of its formulation and quality controls.

Tools & Resources

Selection Charts

FAQs & Troubleshooting

FAQs

  1. What reaction conditions were used to define Authenticase®?
  2. How does Authenticase® improve the quality and fidelity of PCR gene assembly?
  3. How do I convert my gene of interest into oligonucleotides?
  4. Why do you recommend setting up two tubes for the PCR reaction containing different amounts of Authenticase®-treated samples as templates?
  5. Can I use Authenticase® for genome editing applications?
  6. Does Authenticase® recognize single base pair mismatches or indels (insertions/deletions)?
  7. What common additives inhibit Authenticase®?
  8. What PCR reagents are recommended for DNA amplification in genome editing (CRISPR/Cas9, TALEN, ZFN) mismatch detection assays?
  9. Can I use Authenticase® genome editing (CRISPR/Cas9, TALEN, ZFN) mismatch detection assays with unpurified PCR products?
  10. What size PCR amplicon should I design to analyze the genomic editing efficiency?
  11. If my PCR reaction yield is low, can I add more than 5 µl of the PCR reaction to the digestion reaction?
  12. Why do I see an extra band when I run the undigested heteroduplex on a Bioanalyzer or agarose gel?
  13. What are the differences between Mismatch Endonuclease I (NEB #M0678) and Authenticase (NEB #M0689)?

Quality, Safety & Legal

Quality Assurance Statement

Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.

Specifications

The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]

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.

Legal and Disclaimers

Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email busdev@neb.com.

This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.

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.

Other Products You May Be Interested In