DNA Cloning
Gene Cloning : Ability to construct recombinant DNA molecules and maintain them in cells
1. Foreign DNA is digested with a restriction enzyme
2. Bacterial plasmid is cut with the same restriction enzyme
3. A fragment of DNA is inserted into the plasmid DNA using DNA ligase to form a recombinant DNA molecule
4. Transformation: insert recombinant plasmid into bacteria host cells
5. DNA library: Each host cell contain different DNA fragment from the original organism (hybrid molecules)
6. Cells plated on agar medium and grow colonies
1. Isolation of DNA
- Lyse the cell: open PM
- Remove membrane lipid: detergent (SDS)
- Remove proteins: protease
- Remove RNA: RNase
- Precipitate DNA: alcohol
2. Digest DNA with Restriction Enzymes (endonucleases)
- DNA fragmented
- Incubation with buffer + restriction enzymes
- EcoRI, HindIII
3. Plasmid cut with same restriction enzymes
- Plasmid is a vector that provides the information necessary to propagate the cloned DNA in the replicating host cell
- Structure
→ Origin of replication: allows DNA to replicate independently of the chromosome of the host
→ MCS (multiple cloning site): Restriction enzyme recognition only occurs here. Allow DNA fragment to be inserted at a specific point within the
vector so that the DNA insertion does not occur at the Origin or AB resistance gene and disrupt their function
→ Antibiotic Resistance gene
- Sticky or Blunt ends created by restriction enzymes
→ EcoRI generates protruding 5'ends that are complementary to each other, enabling annealing
- Different DNA pieces cut with the same restriction enzymes can recombine into the plasmid
- Sticky ends anneal via H-bonds until DNA ligase acts on ligation
Cloning Strategy
- Single restriction enzyme
→ Non-directional, so 4 colonies picked
→ Self-ligation of the vector occur: vector reforms the circle withouth the DNA added. This has a higher chance to occur than the ligation of the
foreign DNA, so most of the time self-ligation occurs
→ Alkaline phosphatase: removes phosphate to prevent self ligation, but it might remove phosphate from DNA
- 2 Restriction enzymes
→ MCS in plasmid vector and insert DNA is cut using 2 different chosen restriction enzymes eg) HindIII, KpnI
→ Production of 2 complementary overhang, preventing self-ligation and allow DNA insertion onto the plasmid
- Blunt-End Cloning
→ no overhang bases at the terminus of the plasmid, so occurs 100X slower than sticky end ligation
→ fewer sequence limitation between the insert DNA and the plasmid vector
4. DNA ligation
- Inactivate restriction enzyme by raising temperature
- DNA ligase + ATP + insert DNA + alkaline phosphatase : seal the nicks to reform the circle at the 2 DNA ends
- Catalyse formation of phosphodiester bond
- Add excess DNA fragments relative to the plasmid so that majority of the plasmids will reseal with DNA added
Ligation Methods
- DNA topoisomerase I
→ Topoisomerase I pecifically recognise and digest DNA sequence CCTT and unwinds the DNA and religates it again at the 3' phosphate group of the
last thymine
→ TOPO vectors carry CCTT sequence at both ends
→ Topoisomerase I catalyse ligation
- DNA recombinase
→ Cloning by homologous recombination
→ PCR is preformed with primers with overlapping sequences with the vectors
→ DNA recombinase recognises the overlapping sequences and catalyse recombination of the PCR product into the vector
Bacterial Transformation
Methods
- Electroporation: Recombinant DNA inserted into the bacterial cell through pores created by an electrical field
- CaCl2: The bacterial cells become competent to accept foreign DNA when incubated with CaCl2 in cold condition, as the cell surface becomes more
permeable to DNA
Gene Cloning : Ability to construct recombinant DNA molecules and maintain them in cells
1. Foreign DNA is digested with a restriction enzyme
2. Bacterial plasmid is cut with the same restriction enzyme
3. A fragment of DNA is inserted into the plasmid DNA using DNA ligase to form a recombinant DNA molecule
4. Transformation: insert recombinant plasmid into bacteria host cells
5. DNA library: Each host cell contain different DNA fragment from the original organism (hybrid molecules)
6. Cells plated on agar medium and grow colonies
1. Isolation of DNA
- Lyse the cell: open PM
- Remove membrane lipid: detergent (SDS)
- Remove proteins: protease
- Remove RNA: RNase
- Precipitate DNA: alcohol
2. Digest DNA with Restriction Enzymes (endonucleases)
- DNA fragmented
- Incubation with buffer + restriction enzymes
- EcoRI, HindIII
3. Plasmid cut with same restriction enzymes
- Plasmid is a vector that provides the information necessary to propagate the cloned DNA in the replicating host cell
- Structure
→ Origin of replication: allows DNA to replicate independently of the chromosome of the host
→ MCS (multiple cloning site): Restriction enzyme recognition only occurs here. Allow DNA fragment to be inserted at a specific point within the
vector so that the DNA insertion does not occur at the Origin or AB resistance gene and disrupt their function
→ Antibiotic Resistance gene
- Sticky or Blunt ends created by restriction enzymes
→ EcoRI generates protruding 5'ends that are complementary to each other, enabling annealing
- Different DNA pieces cut with the same restriction enzymes can recombine into the plasmid
- Sticky ends anneal via H-bonds until DNA ligase acts on ligation
Cloning Strategy
- Single restriction enzyme
→ Non-directional, so 4 colonies picked
→ Self-ligation of the vector occur: vector reforms the circle withouth the DNA added. This has a higher chance to occur than the ligation of the
foreign DNA, so most of the time self-ligation occurs
→ Alkaline phosphatase: removes phosphate to prevent self ligation, but it might remove phosphate from DNA
- 2 Restriction enzymes
→ MCS in plasmid vector and insert DNA is cut using 2 different chosen restriction enzymes eg) HindIII, KpnI
→ Production of 2 complementary overhang, preventing self-ligation and allow DNA insertion onto the plasmid
- Blunt-End Cloning
→ no overhang bases at the terminus of the plasmid, so occurs 100X slower than sticky end ligation
→ fewer sequence limitation between the insert DNA and the plasmid vector
4. DNA ligation
- Inactivate restriction enzyme by raising temperature
- DNA ligase + ATP + insert DNA + alkaline phosphatase : seal the nicks to reform the circle at the 2 DNA ends
- Catalyse formation of phosphodiester bond
- Add excess DNA fragments relative to the plasmid so that majority of the plasmids will reseal with DNA added
Ligation Methods
- DNA topoisomerase I
→ Topoisomerase I pecifically recognise and digest DNA sequence CCTT and unwinds the DNA and religates it again at the 3' phosphate group of the
last thymine
→ TOPO vectors carry CCTT sequence at both ends
→ Topoisomerase I catalyse ligation
- DNA recombinase
→ Cloning by homologous recombination
→ PCR is preformed with primers with overlapping sequences with the vectors
→ DNA recombinase recognises the overlapping sequences and catalyse recombination of the PCR product into the vector
Bacterial Transformation
Methods
- Electroporation: Recombinant DNA inserted into the bacterial cell through pores created by an electrical field
- CaCl2: The bacterial cells become competent to accept foreign DNA when incubated with CaCl2 in cold condition, as the cell surface becomes more
permeable to DNA
