Week 4 Flashcards
Site-directed mutagenesis (SDM) basics
Overlap extension w/ complementary sequence and w/ 2 PCRs. One primer upstream,another downstream.
Oligonucleotide directed mutagenesis basics
Primers going both directions. Unmethylated + mutant DNA only remains.
Reverse genetics (1 point,1 note)
Block or alter action of targeted gene
NOTE can activate other genes
Homologous Recombination (how?)
Repair of double strand breaks (DSB). 5’ digestion, 3’ invasion by sister chromatid.
Homologous Recombination (why?)
Modify selected genes or replace regions of chromosome
PCR verification
Amplify product
Can identify orientation and accidental deletions
Nuclease activity
Random place w/ double strand breaks (DBS).
Allows in vivo DNA editing
Nonhomologous end joining
Insertion, deletion, etc. allowed
Homology directed repair
Put in donor DNA, incorporatedas DSB repairs
Zinc Finger Nuclease basics
Adaptable to structure, therefore selectable. 2 needed (leaky), no open source collection and may bind to neighbouring DNA.
TALENS (transcription activator-like effector nucleases)
Lots of aminoacid repeats in each TALE which recognise a single nucleotide. Specific.
CRISPR/Cas (clustered regularly interspaced short palindromic repeats) (6 steps)
- capture of foreign DNA
- insertion of small invaded DNA fragments into CRISPr array (Looks for motifs)
- invader DNA becomes spacer DNA
- Memory fragment for cell
- second infection occurs
- looks for protospaced and cleaves it
CRISPR/Cas uses (3 uses, 1 con)
Stable or transient gene transfer
Commercial libraries available
Specificity by targeting same loci
BUT can mis-target (not unique, due to redundancy in genome)
Transcriptome
Complement of active genes, mRNAs and/or transcripts in a particular tissue at a particular time
Double-stranded RNA (dsRNA or hairpin RNA) (2 points)
- Identified by gene that was on the same loci but on the antisense. Affects phenotype of both.
- Gene silencing
