□ 특징

● CRISPR/Cas9, ZFNs, TALENs을 이용한 genome editing 이후 변이 도입 확인에

● Genome에 도입된 삽입 (Insertion), 결실 (Deletion)을 PCR법으로 쉽게 검출

● 세포로부터 직접 PCR 증폭이 가능하여 신속하고 높은 효율

● Mutation detection에 필요한 모든 제품이 구성된 complete kit

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□ 제품설명

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Guide-itMutation Detection Kit는 zinc-finger nucleases (ZFNs),transcription-activator-like effector nucleases (TALENs), or CRISPR/Cas9으로처리된 cell genome으로부터 PCR 방법으로 삽입또는 결실 변이 (Insertions or deletions: Indel)를 확인할 수 있는 제품이다. 본 제품은 Terra PCR Direct Polymerase Mix(Code 639269)를 사용하여 별도의 DNA 추출 과정 없이 세포로부터target sequence를 증폭할 수 있다. 증폭 산물은 Guide-it Resolvase에 의해 mismatch 서열이 절단되며, 이를 전기영동으로 검출함으로써 유전자 변이을 쉽게 확인할 수 있다. 그림 1. TheGuide-it Mutation Detection Kit is used to confirm the presence of mutations ingenomic DNA. In the first step your target sequence is amplifieddirectly from your target cells using the Terra PCR Direct Polymerase includedin the kit, so there is no need to extract and purify genomic DNA from yourcell population prior to amplification of your target sequence. The amplicon isthen melted and hybridized to form the mismatched targets that can be cleaved by the Guide-it Resolvase.그림 2. Comparisonof the Guide-it and Surveyor assays for detecting mutations in mammalian cells. 293T cells weretransfected with plasmids encoding Cas9 and a sgRNA specific for the AAVS1 locus. Transfected cells were harvested 48 hr post-transfection and mixed withuntransfected cells at varying ratios. An amplicon containing the targeted AAVS1 locus was generated using Terra Direct Polymerase Mix and the PCR products werepurified and cleaved using either Guide-it Resolvase or the Cel1 enzyme(Surveyor assay). Mutations were easily discernable when using the Guide-itkit. In contrast, the Surveyor Assay showed considerable smearing, making itdifficult to determine cleavage efficiency and reducing the ability to detectlow levels of mutation.

□ 적용

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□ 구성품

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□ 보존

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-20℃