Determining Genome Targeting Efficiency using T7 Endonuclease I (M0302 )

Protocols.io also provides an interactive version of this protocol where you can discover and share optimizations with the research community. 

EnGen Mutation Detection Kit (NEB #E3321 ) Now Available

Overview:

T7 Endonuclease I recognizes and cleaves non-perfectly matched DNA. This protocol describes how to determine genome targeting efficiency by digesting annealed PCR products with T7 Endonuclease I. In the first step PCR products are produced from the genomic DNA of cells whose genomes were targeted using Cas9, TALEN, ZFN etc. In the second step, the PCR products are annealed and digested with T7 Endonuclease I. Fragments are analyzed to determine the efficiency of genome targeting.

Required Materials:

  • Q5® Hot Start High-Fidelity 2X Master Mix (M0494)
  • T7 Endonuclease I (M0302)
  • 0.25 M EDTA
  • Purified genomic DNA from targeted cells
  • PCR primers to amplify a ~1 kb region containing the target site
    • The target site should be offset from the center of the amplicon so that digestion produces easily resolvable DNA fragments
    • PCR primer design is critical. Please visit NEB's Tools and Resources page to optimize your primer design using the NEB Tm Calculator
  • A PCR thermocycler with programmable temperature ramp rate
  • DNA purification system - we recommend Ampure XP beads
  • Apparatus to quantitate DNA - spectrophotometer or fluorometer
  • Apparatus to analyze DNA fragments – e.g. Agilent Bioanalyzer, Qiagen Qiaxel, or standard agarose gel electrophoresis

PCR:

  1. Set up a 50 µl PCR reaction using ~100 ng of genomic DNA as a template. For each amplicon set up 3 PCR reactions using the following templates
    1. gDNA from targeted cells (e.g. Cas9, or TALEN transfected cells)
    2. gDNA from negative control cells (e.g. non-specific DNA transfected cells)
    3. water (i.e. no template control)

    PCR using Q5 High-Fidelity DNA Polymerase
    Component 50 µl reaction Final Concentration
    Q5 Hot Start High-Fidelity 2X Master Mix (M0494) 25 µl 1X
    10 µM Forward Primer 2.5 µl 0.5 µM
    10 µM Reverse Primer 2.5 µl 0.5 µM
    Template DNA variable 100 ng total
    Nuclease-free water To 50 µl

  2. Gently mix the reaction. Collect all liquid to the bottom of the tube by a quick spin if necessary. Transfer PCR tubes to a PCR machine and begin thermocycling.

  3. Cycling Conditions
    Step Temperature Time
    Initial Denaturation 98°C 30 seconds
    35 cycles

    98°C 5 seconds
    *50-72°C 10 seconds
    72°C 20 seconds
    Final Extension 72°C 2 minutes
    Hold 4-10°C
    *Use of the NEB Tm Calculator is highly recommended.

    Note: Q5 Hot Start High-Fidelity 2X Master Mix does not require a separate activation step. Standard Q5 cycling conditions are recommended.
  4. Analyze a small amount of the of the PCR product to verify size and appropriate amplification.
  5. Purify the PCR reaction using 90 µl of Ampure XP beads following the manufacturer’s recommendations. Other PCR purification systems (e.g. Monarch PCR & DNA Clean Up Kit, or Zymo DNA Clean and Concentrator™) are acceptable.
  6. Elute PCR products in 30 µl of water, recovering 25 µl.
  7. Measure the concentration of the purified PCR products.

T7 Endonuclease I digestion:

  1. Assemble reactions as follows

  2. Component 19 µl annealing reaction
    DNA 200 ng
    10X NEBuffer 2 2 µl
    Nuclease-free Water To 19 µl

  3. Anneal the PCR products in a thermocycler using the following conditions:

  4. Hybridization Conditions
    Step Temperature Ramp Rate Time
    Initial Denaturation 95°C 5 minutes
    Annealing
    95-85°C -2°C/second
    85-25°C -0.1°C/second
    Hold 4°C Hold

  5. Add T7 Endonuclease I to the annealed PCR products
  6. Component 20 µl reaction
    Annealed PCR product 19 µl
    T7 Endonuclease I (M0302) 1 µl

    Incubation Time 15 minutes
    Incubation Temperature 37°C

  7. Stop the reaction by adding 1.5 µl of 0.25 M EDTA.
  8. Purify the reaction using 36 µl of Ampure XP beads according to the manufacturer’s suggestion. This step is optional since 1 µl of the reaction will not interfere with analysis on an Agilent Bioanalyzer using DNA1000 reagents.
  9. Elute the DNA fragments in 20 µl of water, recovering 15 µl.

Analysis:

  • Analyze the fragmented PCR products and determine the percent of nuclease-specific cleavage products (fraction cleaved)
  • Calculate the estimated gene modification using the following formula:

  • % gene modification = 100 x (1 – (1- fraction cleaved)1/2)

    Guschin, D.Y., et. al.(2010) A rapid and general assay for monitoring endogenous gene modification. Methods Mol Biol, 649, 247–256.