What is the function of zinc finger nucleases?
Zinc finger nucleases (ZFNs) are a class of engineered DNA-binding proteins that facilitate targeted editing of the genome by creating double-strand breaks in DNA at user-specified locations.
How do you clone a plasmid DNA?
The basic steps are:
- Cut open the plasmid and “paste” in the gene. This process relies on restriction enzymes (which cut DNA) and DNA ligase (which joins DNA).
- Insert the plasmid into bacteria.
- Grow up lots of plasmid-carrying bacteria and use them as “factories” to make the protein.
What is cloning into a plasmid?
In its simplest form, PCR based cloning is about making a copy of a piece of DNA and at the same time adding restriction sites to the ends of that piece of DNA so that it can be easily cloned into a plasmid of interest.
How does a zinc finger nuclease find its target within a genome?
Zinc-finger nucleases (ZFNs) are artificial restriction enzymes generated by fusing a zinc finger DNA-binding domain to a DNA-cleavage domain. Zinc finger domains can be engineered to target specific desired DNA sequences and this enables zinc-finger nucleases to target unique sequences within complex genomes.
What are the 6 steps of cloning?
In standard molecular cloning experiments, the cloning of any DNA fragment essentially involves seven steps: (1) Choice of host organism and cloning vector, (2) Preparation of vector DNA, (3) Preparation of DNA to be cloned, (4) Creation of recombinant DNA, (5) Introduction of recombinant DNA into host organism, (6) …
How are zinc finger nucleases designed?
Why is CRISPR better than Talen and ZFN?
Recognition of the DNA site in the CRISPR-Cas9 system is controlled by RNA–DNA interactions. This offers many advantages over ZFNs and TALENs, including easy design for any genomic targets, easy prediction regarding off-target sites, and the possibility of modifying several genomic sites simultaneously (multiplexing).
How do zinc fingers interact with DNA?
The zinc fingers recognize specific trinucleotide DNA sequences by insertion of several a-helices in the major groove of the DNA. The CCHH domains are organized in tandem, and the cooperative binding of a-helices contribute to the strength and specificity of the protein-nucleic acid interaction.
Why is Crispr better than Talen and Zfn?