제품설명
Guide-it Mutation Detection Kit는 zinc-finger nucleases (ZFNs), transcription-activator-like effector nucleases (TALENs), or CRISPR/Cas9으로 처리된 cell genome으로부터 PCR 방법으로 삽입 또는 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 세포로부터 target sequence를 증폭할 수 있다. 증폭 산물은 Guide-it Resolvase에 의해 mismatch 서열이 절단되며, 이를 전기영동으로 검출함으로써 유전자 변이을 쉽게 확인할 수 있다.



그림 1. The Guide-it Mutation Detection Kit is used to confirm the presence of mutations in genomic DNA. In the first step your target sequence is amplified directly from your target cells using the Terra PCR Direct Polymerase included in the kit, so there is no need to extract and purify genomic DNA from your cell population prior to amplification of your target sequence. The amplicon is then melted and hybridized to form the mismatched targets that can be cleaved by the Guide-it Resolvase.



그림 2. Comparison of the Guide-it and Surveyor assays for detecting mutations in mammalian cells. 293T cells were transfected with plasmids encoding Cas9 and a sgRNA specific for the AAVS1 locus. Transfected cells were harvested 48 hr post-transfection and mixed with untransfected cells at varying ratios. An amplicon containing the targeted AAVS1 locus was generated using Terra Direct Polymerase Mix and the PCR products were purified and cleaved using either Guide-it Resolvase or the Cel1 enzyme (Surveyor assay). Mutations were easily discernable when using the Guide-it kit. In contrast, the Surveyor Assay showed considerable smearing, making it difficult to determine cleavage efficiency and reducing the ability to detect low levels of mutation.
적용
구성품 (자세한 내용은 CoA를 참조하세요)
보존
-20℃