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
Authenticase cleaves dsDNA carrying mismatches or indels, leaving either 3′ or 5′ overhangs.
FIGURE 2: Applications of Authenticase
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™
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
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.
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