Authenticase® |NEB酶试剂 New England Biolabs

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产品信息

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® |

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

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™

Authenticase® |

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

Authenticase® |

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.
产品类别:
DNA Repair Enzymes and Structure-specific Endonucleases Products

应用:
Genome Editing Applications

  • 产品组分信息

    本产品提供以下试剂或组分：

    NEB # 名称 组分货号 储存温度 数量 浓度
    • 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

  • 特性和用法

    反应条件

    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)

    贮存溶液

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

    热失活

    否

  • 优势和特性

    Features

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

  • 相关产品

    相关产品

    • Q5® 热启动超保真 2X 预混液
    • M0568 Thermolabile Exonuclease I
    • 快速 CIP
    • Monarch® PCR & DNA 纯化试剂盒（5 μg）
    • PCR Marker
    • OneTaq® Quick-Load® 2X 预混液（提供标准缓冲液）
    • Nuclease-free Water
    • NEBuilder® 高保真 DNA 组装克隆试剂盒

操作说明、说明书 & 用法

  • 操作说明

    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)

  • 说明书

    产品说明书包含产品使用的详细信息、产品配方和质控分析。

    • manualM0689

工具 & 资源

  • 选择指南

    • Activities of DNA Repair Enzymes and Structure-specific Endonucleases
    • Properties of DNA Repair Enzymes and Structure-specific Endonucleases

FAQs & 问题解决指南

  • 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)?