BIO282 Lecture Notes - Lecture 33: Bamhi, Plasmid, Dna Polymerase
Learning objectives
By the end of the lecture you should understand:
1. The principle of Genetic Engineering
2. The process overview
3. Target gene amplification
4. Cloning using restriction/ligation
5. Which vectors can be used to transfer genes
6. The steps to genetically engineer bacteria/plants/animals
1. Genetic Engineering principle
Genetic Engineering is the modification of an organisms genome using DNA from
another organism
This provides a means to shift a desirable trait from one organism into another that
doesn’t have it
Organisms created in this way are called Genetically Modified Organisms (GMOs) or
recombinant organisms or simply recombinants.
2. Genetic Engineering overview
• The process in essence is to take a particular target DNA fragment, amplify it, and then
treat it in a way that you can then clone it and select it
• Target gene amplification-PCR can be used to generate the target gene for
cloning
Phusion DNA Polymerase has been engineered to bring together a novel Pyrococcus-like
enzyme with a processivity- enhancing domain.
This generates PCR products with accuracy and speed previously unattainable with a
single enzyme
Restriction and ligation are essential for the cloning process
• So the target DNA has been amplified, now it needs to be prepared so it can be
cloned
• To cut the DNA this is done in a number of ways
o Need to identify restriction sites in your DNA sequence itself (so when you
amplify it the restriction sites are already there)
o Need to build them into your priming sites so that when your product is
amplified you can cut that product and generate your restriction fragment
• Typically you need to prepare your DNA so it can be cloned into your desired
vector
• When you're trying to clone a gene or fragment, you need to treat both ends of
the fragment so that the ends are complementary
Document Summary
Genetic engineering is the modification of an organisms genome using dna from another organism. This provides a means to shift a desirable trait from one organism into another that doesn"t have it. Phusion dna polymerase has been engineered to bring together a novel pyrococcus-like enzyme with a processivity- enhancing domain. This generates pcr products with accuracy and speed previously unattainable with a single enzyme. Straight forward bamhi cloning (only 1 enzyme) can result in a number of different products: your gene could go in a reverse orientation producing a undesired response (doesn"t switch a gene on) If you use two enzymes you can treat your dna to have a specific orientation in respect to complementary binding. Directionality is important for expression can switch a gene on: dna is transferred via vectors. Ligated dna is cloned into a vector (plasmid, virus) A process is required to select for the introduced gene in the host cell 10.
