New England Biolabs, M0689L, Authenticase®

Mixture of structure-specific nucleases capable of recognizing and cleaving outside mismatches and indels Related Categories Validation of CRISPR-based Gene Editing,, DNA Repair Enzymes and Structure-specific Endonucleases Specification 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 FAQ Q: What reaction conditions were used to define Authenticase®? A: A reaction is defined as 1 µl of Authenticase in a 20 µl reaction containing 1X Authenticase Reaction Buffer, a mixture of 0.33 pmol of 60-mer heteroduplex of A/C, T/G and 2 bp indel (insertion and/or deletion) mismatches, respectively. After incubation at 42°C for 30 min, >90% of the substrate DNA was digested, as determined by 4% agarose E-gel. More details of the Authenticase protocol and usage can be found in the product manual. Q: How does Authenticase® improve the quality and fidelity of PCR gene assembly? A: 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 base pairs (bp) on double-stranded DNA. By enzymatically removing “mistakes” prior to the final renaturation and amplification step, Authenticase can reduce errors in oligo-based PCR gene assembly. Q: How do I convert my gene of interest into oligonucleotides? A: While NEB® does not offer a web tool to design oligos for gene synthesis, many 3rd party offerings exist. For example, DNAWorks (https://hpcwebapps.cit.nih.gov/dnaworks/) automates the design of oligonucleotides for gene synthesis by PCR-based methods. Q: Why do you recommend setting up two tubes for the PCR reaction containing different amounts of Authenticase®-treated samples as templates? A: DIY gene synthesis workflows often enrich the full-length target gene prior to DNA assembly or cloning into the holding vector. We do not require the user to set up multiple enrichment reactions; however, since amplification by PCR can produce variable amounts of the full-length target, we suggest establishing more than one PCR reaction to ensure a sufficient amount of the full-length target is generated. After PCR, select the amplicon with the higher purity by running an agarose gel analysis or Bioanalyzer prior to cloning the fragment. Q: Can I use Authenticase® for genome editing applications? A: Yes. Authenticase can be used to detect mismatch/indel formation (on-target editing events) in genome editing workflows. It has been formulated to efficiently cleave mismatches in heteroduplex DNA and outperforms traditional T7 Endo I screening across a broad range of mutation/WT ratios. Please see Mismatch Detection Assay (NEB #M0689). Q: Does Authenticase® recognize single base pair mismatches or indels (insertions/deletions)? A: The answer is complex. There are eight types of single base mismatches: C/C, T/C, A/C, T/G, A/G, G/G, T/T and A/A. Authenticase can recognize 7 of the 8 mismatches. The exception is an A/G mismatch. Cleavage of these mismatches using Authenticase is more efficient than with T7 Endonuclease I, which has limited activity on these single-base mismatches (Biochemistry 2004, 43, 4313-4322). Both enzymes have no problem cleaving heteroduplex DNA with 1-10 bp Indels or 2-10 bp mismatches. Q: What common additives inhibit Authenticase®? A: A chelating agent, such as EDTA, will inhibit the reaction. Please note, Authenticase requires Mg2+, which is in the reaction buffer. Q: What PCR reagents are recommended for DNA amplification in genome editing (CRISPR/Cas9, TALEN, ZFN) mismatch detection assays? A: NEB recommends Q5® Hot Start High-Fidelity 2x Master Mix (NEB #M0494). Q5 DNA Polymerases offer high fidelity and robustness. For some highly repetitive regions, LongAmp® Taq DNA Polymerase (NEB #M0323) has worked well. Q: Can I use Authenticase® genome editing (CRISPR/Cas9, TALEN, ZFN) mismatch detection assays with unpurified PCR products? A: Yes. For quick analysis, we recommend using 6 μl of the appropriate PCR reaction directly in a 20 µl Authenticase reaction when the following PCR reagents are used: Q5® Hot Start High-Fidelity 2x Master Mix (NEB #M0494) or OneTaq DNA Polymerase (NEB #M0480). Alternatively, one can use 1-12 μl of DNA processed by Exo-CIP™ Rapid PCR Cleanup Kit (NEB #E1050) directly in a 20 µl Authenticase reaction when the following PCR reagents are used: Q5 High-Fidelity DNA Polymerase (NEB #M0491) with Q5 Reaction Buffer (up to 6 µl) or OneTaq DNA Polymerase Reaction Buffer (up to 12 µl). Additional details are in the product manual. For a more accurate estimation of genomic editing efficiency, amplicons purified from Monarch® PCR & DNA Cleanup kit (NEB #T1030) are recommended. Q: What size PCR amplicon should I design to analyze the genomic editing efficiency? A: If a Bioanalyzer will be used to analyze the genome editing efficiency, NEB recommends designing PCR products around 700 bp with anticipated sizes of cleaved products to be approximately 450 and 250 bp. Q: If my PCR reaction yield is low, can I add more than 5 µl of the PCR reaction to the digestion reaction? A: The digestion has been optimized for use with up to 5 µl of unpurified PCR reaction. Adding more than 5 µl of the PCR reaction could interfere with the Authenticase digestion reaction. We recommend optimizing the PCR reaction to get a higher yield or concentrating the PCR reaction by column purification using NEB Monarch PCR & DNA Cleanup Kit (5 μg) (NEB #T1030). Q: Why do I see an extra band when I run the undigested heteroduplex on a Bioanalyzer or agarose gel? A: In some cases, undigested heteroduplex DNA will run slightly slower than the homoduplex DNA. Depending on the composition of the mutations, this can sometimes be seen as a band running above the homoduplex on an agarose gel. Q: What are the differences between Mismatch Endonuclease I (NEB #M0678) and Authenticase (NEB #M0689)? A: Mismatch Endonuclease I only cleaves the following DNA mismatches: G/G, G/T and T/T. Authenticase will cleave all single base mismatches (except for G/A) and all 2+ base pair DNA mismatches and indels (insertion and/or deletion).