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Cat. No.: GEMD-25 (for 25T)

Cat. No.: GEMD-100 (for 100T)

Description

Genome-Editing Mutation Detection Kit, also known as the Gene-Editing Mutation Detection Kit, is a simple, reliable, and fast kit used to detect mutations in genomic DNA after gene editing. The kit is based on the T7 Endonuclease I (T7EI) enzyme, which can recognize and cleave incompletely matched DNA double strands (heteroduplex DNA).

The kit includes a comprehensive set of reagents, including one-step genomic DNA extraction, high-fidelity PCR amplification, denaturation, annealing, and T7EI digestion. It also provides a positive control.

The kit is designed for convenient use. The one-step genomic DNA extraction is rapid and convenient, and the extracted DNA can be directly used for high-fidelity PCR amplification. The PCR products can then be used directly for denaturation, annealing, and T7EI digestion without the need for additional purification steps.

The principle of the kit for detecting genomic editing mutations is as follows: The cells, tissues, or organs to be edited are transfected or infected with nucleases such as CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 nucleases), TALEN (Transcription activator-like effector nucleases), or ZFN (Zinc-finger nucleases). Under the intervention of cellular repair mechanisms, these nucleases will produce insertions or deletions (indels) at specific loci in the genome guided by specific nucleic acids such as guide RNA (sgRNA). After extracting the corresponding genomic DNA, the edited DNA fragments are amplified using high-fidelity PCR. The amplified target fragments are denatured, annealed, and then subjected to T7EI digestion. If successful gene editing resulting in insertions or deletions occurs in some cells, incomplete base pairing will occur between the unedited and edited genes or between different mutated genes in the denatured and annealed PCR products. T7EI can recognize and cleave the incompletely matched double-stranded DNA. By analyzing the T7EI digestion products through gel electrophoresis, it is possible to determine whether the genome has been successfully edited and the editing efficiency.

The kit provides a one-step genomic DNA extraction reagent, which allows for DNA extraction with a single incubation step.

The kit includes a set of control templates and primer premix (Control Template & Primers) for use as positive controls. The control template contains both unedited and edited gene fragments. The length of the unedited gene fragment after PCR amplification is 525bp, while the edited gene fragment contains a 7-base pair deletion mutation, resulting in a PCR product of 518bp. After PCR amplification, denaturation, and annealing, the control template forms incompletely matched double-stranded DNA. When digested with T7EI, the 518bp fragment is cleaved into two different-sized fragments, 288bp and 230bp, while the completely matched double-stranded DNA remains intact and is not cleaved by T7EI. Gel electrophoresis allows for clear visualization of the uncut 525bp and 518bp bands, as well as the cut 288bp and 230bp bands. Typically, the 525bp and 518bp bands appear as a single band due to their very similar molecular weights.

The PCR products obtained from this kit can be directly used for denaturation, annealing, and T7EI digestion. The PCR amplification system of the kit is compatible with T7EI digestion. After PCR, there is no need for purification of the PCR products. They can be directly subjected to denaturation, followed by the addition of T7EI enzyme for digestion and identification analysis. In addition, the digestion of incompletely matched double-stranded DNA by T7EI can be completed in just 15 minutes.

Precautions

• When preparing the cell lysis solution, it is recommended to use the DNA Extraction Solution and Enzyme Mix mixture as soon as possible after thorough mixing. Leaving it for too long may affect the efficiency of DNA extraction.
• For first-time users of this kit, it is strongly recommended to use the Control Templates & Primers provided in the kit as positive controls. After denaturation and annealing of their PCR products, they can be used as positive controls for T7 Endonuclease I enzyme digestion.
• This product is intended for scientific research purposes by professionals only and should not be used for clinical diagnosis or treatment. It should not be used for food or drug purposes and should not be stored in residential areas.
• For your safety and health, please wear laboratory attire and disposable gloves when handling the kit.

Storage

The minimum shelf life is 1 year at -20°C. Avoid multiple freeze-thaw cycles.