RESUMO
The indel-forming non-homologous end joining (NHEJ) pathway repairs double strand breaks in mammalian genomes, resulting in mutation formation following genome editing. Common techniques employed to identify these mutations include the amplified fragment length polymorphism (AFLP) and SURVEYOR assays, which are time consuming, laborious, and only offer a low level of sensitivity. An alternative to these approaches, which is examined in this study, is based on the quantitative PCR high-resolution melting (qPCR-HRM) curve analysis technique and offers simple implementation, is capable of handling large sample sizes, takes no more than 90 min, and produces sensitive results. Using the newly discovered RNA-guided CRISPR/Cas systems, the IL2RG and EMX1 genes were edited in the human 293T cell line in order to compare the mutation detection accuracies of the aforementioned methods. Genomic mutations were simulated by mixing mutated DNA fragments with normal fragments along a concentration gradient. The results of this comparative study showed that the HRM approach was both reproducible and accurate.
Assuntos
Análise do Polimorfismo de Comprimento de Fragmentos Amplificados/métodos , Sistemas CRISPR-Cas/genética , Análise Mutacional de DNA/métodos , Genoma/genética , Mutação/genética , Animais , Linhagem Celular , DNA/análise , DNA/genética , Células HEK293 , Humanos , Camundongos , Alinhamento de SequênciaRESUMO
Using immunofluorescence staining, the patterns of histone H3 K4m2 of metaphase X chromosomes were measured in cloned cattle derived from FFB and FOV donor cells. The results demonstrated that the modification pattern of H3 K4m2 in the ear tissue of all clones was largely consistent with that of the donor cell line FFB and conventionally produced cattle, but different from that of the donor cell line FOV.
Assuntos
Metilação de DNA , Imunofluorescência/métodos , Histonas/metabolismo , Lisina/metabolismo , Cromossomo X/metabolismo , Animais , Bovinos , Células Clonais , Embrião de Mamíferos/citologia , Epigênese Genética , Feminino , Fertilização in vitro , Histona Desacetilases/metabolismo , Histonas/genética , Lisina/genética , Cromossomo X/genéticaRESUMO
In recent years, geneticists have been exploring the less visible parts of genome more thoroughly. They are coming to realize that these much more layers of genomic information, distinct from the protein-coding genes, connect in surprisingly deep and potent ways to growth and development in the high eukaryotes. This article reviews a number of startling observations about the extent of non-protein-coding RNA transcriptions, DNA methylation and covalent histone modifications in the complex organisms.