BIOS 452 Lecture Notes - Lecture 22: Restriction Enzyme, Mutation, Transcription Activator-Like Effector Nuclease
Document Summary
Site directed mutagenesis- introduced by chemically synthesized primers in the. Plasmid> denature and anneal oligonucleotide primers with mutation-> use dna polymerase to extend and incorporate mutagenic primers-> pcr amplification(intro muation)-> dpnl digestion(eliminate methylated parent wild type dna). Pcr amp- digest non mutated parental dna template with methylation specific nuclease and anneal new strands. Genome editing- dna inserted, replaced or removed using artificially engineered nucleases. Nucleases create site-specific ds breaks, use endogenous mechanisms to repair by homologous recombination and nonhomologous end joining. 4 fam of engineered nucleases: zinc finger nucleases, hybrid meganucleases, talens, crispr/cas. Delete- crispr/cas9 typeii restriction endonucleases- cleave dnas at specific base seq(cut) Dna ligase- join 2 dna molecules or fragments. Dna pol i0 fills gaps in duplexes by addition of nucleotieds to 3" end. Reverse transcriptase- make dna copy of an rna molecule(copy from a mrna template) Polynucleotide kinase- adds a phosphate to the 5"-oh end of a polynucleotide to label it or permit ligation(mark)
