Genetic engineering 3 Flashcards
What is the polymerase chain reaction used for?
Used to amplify minute amounts of DNA by repeated cycles of in vitro DNA replication
In PCR how does DNA amplification proceed?
In an exponential scale
What are the 3 purposes of PCR?
1) Detection of infectous agents and diseases
2) Ampilification of limited mounts of DNA
- To obtain enough for cloning or research
- Able to see small differences more easily
3) Modification (insert point-mutations and restiction sites)
What are the 5 requirments in PCR?
1) Very small amount of DNA to be amplified (the template)
2) Knowledge of the exact DNA sequence at the START and END of the sequence to be amplifies
- In order to make 2 DNA primers (double strand)
3) Taq polymerase
- A DNA polymerase
4) Lots of dNTPs
5) Thermal cycler
- Machine that can hold reaction tubes at different temperatures for precise times
What are the stages of PCR?
1) DNA denaturation at 95 degrees
- Break H bonds
2) Annealing of primers to template DNA at 55 degrees
- Anneal at 3’ end of template strand
3) Elongation of primers until the end of the template at 72 degrees
- Taq polymerase binds to primer and elongates it
- Continues until it falls off template at the end
4) Product from previous cycle become s the template for the next cycle
- Exponential increasee
Why need to add the primer to the 3’ end of the template strand?
- To allow DNA synthesis of the other strand in the 5’ to 3’ direction
- As DNA is anti-parallel
What are the 2 ways primers can be used to change the DNA in PCR?
1) Use primers to insert mutations
- Primers are incorporated into the final PCR product
2) Primers can be used to add suitable restriction sites and enable DNA engineering
Why are primers used to add restriction sites?
- To enable subsequent cloning of the product
- Able to remove fragment of DNA from a large DNA strand in order to clone it
- Unable to do this without a restriction site
What is DNA cloning?
- Isolating DNA of interest and maintaining it outside of it’s biological context
- Then, generating an unlimited supply of it
- Involves inserting a piece of DNA into a host DNA molecule
What is a DNA vector and how are they used?
- Host DNA molecule which can be fused with a insert and confer its properties to the insert
- DNA which can be maintained and replicated naturally by the host
- Usually a plasmid
What is used to ‘cut’ and ‘stick’ an insert into a vector?
Restriction enzymes
What are examples of good host organisms and why?
Bacteria and viruses
As they:
- Have short life spans
- Are easy and cheap to grow in vast quantities
- Easy to break up and purify DNA from them
What are plasmids?
- Most common type of vector
- Small, circular DNA found naturally in bacteria
- Not essential for the bacteria to live
- Separate from the bacterial chromosomes
- Give extra benefits to the organism such as antibiotic resistance
What are the 3 essential properties of plasmids to enable DNA cloning?
1) Contain origin of DNA- replication (Ori)
- Allow to exist and replicate independently of the chromosome
2) Contain antibiotic resistance gene
- Can select for bacteria containing it
3) Have recognition sites to facilitate insertion of DNA
What are the 5 steps of DNA cloning?
1) Generate compatible, cohesive endss in both insert and plasmid by the SAME restriction enzyme
2) Fusion of the insert and the plasmid by DNA ligase
3) Introduce recombinant DNA molecules into bacteria (transformation)
4) Antibiotic selection for bacteria containing plasmids
5) Large scale propagation of bacteria colonies to isolate large amounts of recombinant plasmid
