Techniques 1: Enzymes used in molecular cloning Flashcards
What is recombinant DNA?
A type of DNA sequence made from several sources
What is recombinant DNA used for?
Used to isolate a specific region of DNA that we are interested in
Describe the function restriction enzymes
RE chops up foreign DNA, host DNA is not chopped (due to methylation of restriction sites)
Describe the process of making recombinant DNA
- Initial binding of enzyme is non-specific
- Enzyme moves along until it finds a specific recognition site
- Specific binding triggers structural changes (in enzyme and DNA)
- Catalysis requires Mg2+
- Generates free 5’ phosphates and 3’ -OH ends
- Sticky ends bind together (vector and insert must be cut from the same/compatible enzyme)
→ This binding is not permanent = no phosphodiester bond - Complementary (sticky) ends have to interact
- Enzymatic reaction = DNA ligase reforms new phosphodiester bonds
Which enzyme can remove the 3’ overhang?
T4 DNA polymerase
Which enzyme can remove the 5’ overhang?
Mung Bean nuclease
What is the most commonly used vector?
A plasmid
What are 3 features common to all vectors?
- Origin of replication (allows plasmid to replicate inside host)
- Selection Marker (survival of host cells that are carrying plasmid)
- Multiple cloning site (unique, where gene of interest is cloned)
What are 2 ways to transfer recombinant DNA to the host?
Electroporation
Chemical transformation (more common)
What is electroporation?
Brief pulse of high voltage
What is Chemical transformation?
Chemically treated
Subject to heat shock
Causes cell membrane changes to allow uptake of DNA
Describe how transformed bacteria can be selected
- Plate bacteria on medium that only allows them to grow if they have taken up the selectable marker gene e.g antibiotic resistance
