Recombinant DNA technologies

What are restriction enzymes?

Type of endonuclease which cleaves phosphodiester bonds

act as bacterial immune system against viruses

needed for DNA cloning and recombinant DNA

What are the types of restriction enzymes?

Type 1 cut remote from recognition sequence

Type 2 cut beside or in recognition sequence

Type 3 cut near recognition sequence

Type 4 cut modified DNA

How do type 2 restriction enzymes function?

Recognition sequences usually palindromic

produce 5 prime/3 prime overhangs or blunt end

products have compatible ends that can be rejoined via DNA ligation forms new phosphodiester bonds (anneal sticky ends)

What is an example of a DNA ligase?

T4 DNA ligase

isolated from T4 bacteriophage, ligates sticky ends requires 5 prime end to be phosphorylated and requires ATP

works at 37 degrees

What are plasmids and why are they used in DNA cloning?

Circular pieces of DNA independent of bacterial chromosomes

have Ori, antibiotic resistant gene (amp) and a multiple cloning site (MCS) which is cluster of RE recognition sites for insertion of DNA fragments

generates more DNA than PCR, can amplify larger fragments, allows expression for production of recombinant DNA

What is directional cloning?

Used to prevent fragment being inserted into plasmid in 2 different orientations

Use 2 different restriction enzymes with different recognition sequences producing different sticky ends at ends of fragment/plasmid

What is an example of the process of transformation?

Mix circular DNA with E.coli on ice for 30mins

heat shock at 37 for 1min

Add LB broth and incubate at 37 for 45mins

Plate E.coli on agar plates with antibiotic

untransformed cells have no resistance so die

How is recombinant DNA used for GH?

Used in treatment for short stature IUGR

Obtained by using recombinant plasmids, which produce human GH protein