CB - Plasmids Flashcards
What is gene cloning?
Gene cloning is the process in which a desirable gene is located and copied out of an organisms DNA
What are the 5 main steps involved in gene cloning?
- The gene is isolated
- The gene is inserted into the vector (Recombinant DNA)
- Transported into a host cell
- Multiplication of recombinant DNA
- Division of host cell
Why may Plasmid vectors be used? (3)
- Generate large amounts of DNA
- Derived from abundant source (Bacteria)
- Can replicate independently of the host cell
What is the difference between Expression vectors and Cloning vectors?
Cloning Vectors- Used to acquire multiple copies of a desirable gene
Expression Vectors- Used to acquire the gene product
What are the 2 main importances of gene cloning?
- To generate large amounts of DNA
- To generate large amounts of protein from your chosen gene
How can gene cloning help in medicine?
- Able to detect mutations that casue genetic diseases
- Able to develop new ways to treat diseases
- Able to develop cures for inherited diseases using Gene Therapy
How is the gene isolated?
PCR
How can PCR be used to isolate a gene?
- Design 2 primers to anneal to either end of the gene
- Lyse the cell
What is a limitation of PCR?
Its difficult to amplify products longer than 10-15Kb
What are restriction enzymes used for?
Used to cut DNA into smaller fragments.
Cuts are made at specific points.
Why don’t restriction enzymes cleave bacterial DNA?
Bacterial DNA contains an ECO RI methylase
What are the 2 types of restriction enzymes?
- Exonucleases- remove nucleotides from the ends
- Endonucleases- break nucleic acid chains in the interior
Whata are the 3 types of restriction endonucleases?
Type I- cuts DNA 1000 bases from a recongition site
Type II- cuts DNA at the recognition site
Type III- cuts DNA 25 bases from the recognition site
