Molecular cloning Flashcards
What is molecular cloning?
A set of experimental methods in molecular biology that are used to assemble recombinant DNA molecules.
What is recombinant DNA?
A sequence of DNA composed of 2 or more different sources - e.g. organisms, synthetic & microorganisms.
What is the purpose of molecular cloning?
To isolate a gene or sequence:
- to analyse mutant genes vs wild type
- to make mutations
- express & purify the proteins
What is a brief overview of molecular cloning?
- make recombinant DNA
- cut & paste vector to the DNA
- put in host & let replication to occur
What is a vector?
- a vehicle to carry your gene
What are 3 main parts of a vector?
- restriction enzyme sites
- origin of replication
- selectable marker
What happens at the restriction enzymes sites?
- where your gene gets cloned (MCS - multiple cloning site)
What happens at the origin of replication?
Independent replication inside the host
What happens at the selectable marker?
survival of host cells that carry plasmid
What are the cons of using a plasmid?
- small (can’t have large insertions)
- low copy number
What 3 things are needed to clone DNA into a vector?
- enzyme
- reagents
- techniques
If a phage grown in one bacterial host failed to grow in a different bacterial host it is said to have been what?
Restricted
If a phage is able to grow in a new host, it is said to have been what?
Modified
What protects DNA from being cut up by a restriction enzyme?
The DNA is methylated
How many classes of restriction enzymes are there?
4
What do type 1&3 restriction enzymes do?
Cleave DNA at random far away from recognition site
What do type 2 restriction enzymes do?
Cut DNA at a defined position
What do type 4 restriction enzymes do?
Cleave modified DNA
What are the 2 types of restriction endings left?
- overhang
- blunt end
What element is required for the catalysis of restriction enzyme once its bound to DNA?
Magnesium
Why would you use different restriction enzymes on a vector?
To prevent the sticking of sticky ends
What is needed to create a phosphodiester bond in the backbone of the nucleotides that have attached to overhanging ‘sticky’ ends?
DNA ligase
How does the DNA ligase mechanism work?
- AMP transferred to enzyme active site.
- AMP transferred to the 5’ Phosphate
- AMP-phosphate bond broken by 3’ OH, forming covalent bond
What is the problem with recombinant DNA?
- There may not be convenient restriction sites
- Not enough DNA
- DNA may be mixed in with other DNA molecules
What occurs if both of the strands of DNA has a phosphate?
Ligation won’t occur
What is the purpose of removing the 5’ phosphate (dephosphorylating the vector)?
Prevent self-ligation of the vector
What is a way to prevent an overhang?
Use a restriction enzyme that leaves a blunt end.
What are the 2 ways of getting recombinant DNA into a host?
- Electroporation
- Chemical transformation
What is the process of electroporation?
Brief pulse of high voltage
What is the process of chemical transformation?
- Chemically treated E. coli
- Subject it to heat-shock
- Causes cell membrane changes that allow uptake of DNA
What is used to kill bacteria that doesn’t have a vector?
Antibiotics
Why is screening necessary after antibiotics have been used to kill other bacteria?
As it isn’t always efficient
