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Authenticase®

Catalog # Concentration Size List Price Quantity Your Price
M0689L 125 reactions $870.00
$783.00
M0689S 25 reactions $218.00
$196.20
Catalog # Size List Price Your Price
M0689L 125 reactions $870.00
$783.00
M0689S 25 reactions $218.00
$196.20
Catalog #
Qty:
 
*On-line ordering is for Canadian customers only. Web pricing is applicable only to orders placed online at www.neb.ca

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.

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.

Reagents Supplied

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration
  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
  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
Features
  • Reduces number of colonies that need to be screened and saves time
  • Can replace T7 Endonuclease I in mismatch detection assay
An exception occurred during the operation, making the result invalid. Check InnerException for exception details.

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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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