Basic genetic engineering Flashcards
What is genetic engineering?
Directly manipulating the genetic material of a hoagie, cell or whole organism for a specific purpose
What is recombinant DNA technology?
The in vitro (outside a cell) and in vivo (inside a cell) techniques used to manipulate nucleic acids which make genetic engineering possible
What end of a DNA strand can nucleotides be added to?
A DNA strand can only be extended by adding a nucleotide to the 3’ OH end
What are the steps for gene cloning? (7)
a) extract DNA from cell
b) cut out the gene
c) cut open plasmid
d) insert the gene (ligase)
e) transform recombinant plasmid into a bacterial host and amplify the clone
f) isolate plasmid DNA from a large no. of bacterial cells
g) Remove cloned gene from the plasmid to use as required
What are the key tools in recombinant DNA technology?
Restriction endonucleases - cut DNA at specific sites
What do bacterial cells use to help protect themselves against foreign DNA entering the cell?
Restriction systems - cut the foreign cell at specific points
Protects its own DNA with restriction and modification enzymes
What is a recognition site?
A short sequence of nucleotides in double stranded DNA compromises a specific recognition site
How does a bacterium protect its own DNA?
Methylates (A) site : GA(A)TTC
How is DNA protected from restriction cutting during DNA replication?
Methylation - restriction enzyme can’t cleave
What are the main types of restriction endonucleases and what do they do? (4)
Type 1 - cut at random position
Type 2 - cut within recognition sites
Type 3 - needs 2 recognition sites to cut
Type 4 - only cut DNA with special chemical modifications
How do type 2 enzymes cut?
They cut in palindromic sequences - each strand have the same sequence when read in the 5’ to 3’ direction
What part of the DNA do type 2 enzymes bind to?
Phosphodiester backbone
What re the 2 types of ends that Type 2 enzymes can make on DNA?
Blunt ends
Sticky ends
What are the key features of a plasmid? (4)
- Circular with multiple copies per cell
- Carry extra genes for specific circumstances
- Some plasmids can be copied from one bacterial cell to another by bacterial sex
- Only some bacterial cells in a species may possess a certain plasmid at any given time
What are the 2 types of plasmids?
- Large (50-100kb), low no. of copies, can transfer cell to cell
- Small (3-10kb), high no. of copies, can’t transfer
What do these terms mean: IS Tn Tra operon OriV OriT
IS = insertion sequence Tn = transposon Tra operon = transfer genes (important) OriV = plasmid origin of DNA replication OriT = origin of transfer
Why is Tra operon important? (2)
It determines the synthesis of a structure called a sex pilus
This brings 2 cells together and triggers plasmid transfer
What is the sex pilus’ role? (4)
- Pilus makes contact and draws the cells together to form a conjugation bridge
- The plasmid DNA is nicked at OriT and rolling circler DNA replication occurs
- The displaced strand is directed into the other cell
- The cells separate each with a plasmid
How do Hfr strains form? (3)
- The plasmid integrates into the bacterial chromosome
- This occurs rarely and is generally at sites of homology provided by IS elements
- This can then lead to transfer of chromosomal DNA from one cell to another
What is the point of plasmid transfer?
Give the host cell a new function that gives it an advantage of other bacteria
Are plasmids smaller or bigger than the chromosome?
Plasmids are smaller
Why is conjugation so important for bacteria?
Antibiotic resistance
What are they key functions of plasmids as vectors? (4)
- based on small naturally occurring non-conjugative plasmids
- replicate autonomously to high copy number in bacteria
- Carry added antibiotic resistance genes
- carry an added multiple cloning site
