Enzymes used in molecular cloning Flashcards
Molecular cloning
Is recombinant DNA (not much use unless we can make more)
Used to isolate specific region of DNA
Overview of molecular cloning
- Cut and paste DNA fragments together using vectors
- Put recombinant DNA into host (E. coli) which acts like a factors
- Selection and host replication, produces lots of progeny, all containing the recombinant DNA
Restriction and modification
You need something to cut the DNA in the right place
Phage grown in one bacterial host failed to grow in a different bacterial host - restriction
Rare progeny phage able to grow in new host
Restriction enzymes
Thousands known and hundreds used
Recognise short, specific DNA sequences
Have 3/4 types
- Type 1 and 3 cleave DNA at random far away from recognition sequence
- Type 4 cleave modified DNA
- Type 2 cut DNA at defined point, either within or near to recognition sequence
Type 2 restriction enzymes
Most widely used ones are protein homodimers
Recognise specific 4-8 bp DNA sequences
DNA sequence often palindromic
Can generate overhangs (5’ or 3’) or blunt ends
Cleavage generates 5’ phosphate and 3’ OH groups
How type 2 restriction enzymes work
- Initial binding is non-specific, Looser, catalytic site not involves so no specific cutting
- Enzymes move along DNA until it finds specific recognition site, can “slide” for short distances, can also jump or hop over long distances if doesn’t encounter specific site
- Specific binding triggers structural changes (enzymes and DNA), recognition specific site -> conformational changes (enzymes and DNA), exact mechanism not yet known
- Catalysis requires Mg2+
- Generates free 5’-phosphate and 3’-OH ends
The ligase reaction
- Complementary “sticky ends” have to interact
- Enzymatic reaction: DNA ligase catalyses the formation of now phosphodiester bonds
DNA ligase mechanism
- AMP is transferred to a lysine residue in enzyme’s active site (from ATP-the cofactor)
- AMP then transferred to 5’-phosphate
- The AMP-phosphate bond attacked by 3’-OH, forming covalent bond and releasing AMP
ATP required to replace AMP used in reaction (i.e. ATP is a cofactor)
Basic molecular cloning
Restriction enzymes cut up DNA and DNA ligase stick it back together
Few problems:
- Might not be convenient restriction sites
- Might not have enough DNA
- My DNA might be mixed with lots of other DNA
Potential problems
Vector has complementary ends - might ligate itself
- Modify vector ends - phosphatase treatment removes 5’ phosphate - no phosphodiester bond can be formed
Gene may inset in wrong orientation
- Use more than one enzymes (also solves first problem) for each end
- PCR
Modifying DNA ends - Addition/removal 5’ phosphate
If only one molecule has phosphate:
- Will get single phosphodiester bond
- Other end will be nicked
- Recognised and repaired in bacterial host
Addition needs T4 Polynucleotide
Removal needs Calf Intestinal Phosphatase (CIP)
Removing DNA overhang
Blunt end cloning might be necessary
Destruction restriction enzyme site
What is a vector
Most commonly used is a plasmid
Essential features, common to all vectors:
1. Origin of replication - independent replication inside host
2. Selectable marker - survival of host cells that are carrying your plasmid
3. Multiple cloning sites - unique sites, where you clone gene
Transfer to host, selection and replication - Transformation
Electroporation
- Brief pulse of high voltage
Chemical transformation
- Chemically treated E.coli
- Subject to heat shock
- Causes cell membrane change that allows take up of DNA
Selection and amplification
Can’t distinguish between your recombinant DNA and e.g. the empty vector
Cloning strategy important
As is screen transformants
