Basic genetic engineering 2 Flashcards
After this lecture you should be able to: Explain the principle of the blue/white selection system used by pUC plasmids Understand how plasmid DNA can be introduced into bacteria and extracted from bacteria Describe the principles of agarose gel electrophoresis and southern blotting Explain what cDNA is and how it can be made
What is a pUC plasmid? (2)
A plasmid with multiple cloning sites
Foreign DNA inserted into multiple cloning sites destroys activity of lacZ gene
What is the lacZ gene and what does it do? (4)
- Allows a-complementation
- LacZ encodes an enzyme (b-galactosidase) that can cleave colourless substrate resulting in galactose and a 5-bromo-4-chloro-indoxyl derivative
- The indoxyl derivative spontaneously dimerises and oxidises to form insoluble blue dye
- Complete lacZ gene encodes the wild-type protein
What happens if there is a mutant lacZ gene?
A mutant lacZ gene lacking the N-terminal 41 AA is not functional but can be made functional by co-expressing there lacZ a-peptide encoded by the lacZ gene in the same cells
What shows up as blue and what shows up as white on an agar plate of lacZ genes?
Blue: E.coli carrying lacZM15 mutation and pUC18 (a-complementation)
White: Recombinant colonies (what you want)
How can you make a functional b-galactosidase enzyme?
a-peptide combined with carboxyl terminals of b-galactosidase
How do we get DNA into bacteria? (3)
Open the membrane pores
- CaCl2 precipitation and heat shock
- Electroporation
- Gene gun (unlikely to use)
What are the common marker genes used to select for plasmid containing bacteria? (6)
- Ampicillin (common)
- Tetracycline (common)
- Chloramphenicol (common)
- Kanamycin
- Bleomycin
- Hygromycin B
How do you isolate plasmid DNA? (9)
- Pick a single colony and grow bacteria in agar overnight
- Lyse bacteria and isolate plasmid DNA
- Bacteria treated with detergent sodium dodecyl sulphate with NaOH to ensure alkaline pH
- SDS disrupts the cell membrane and denatures proteins as well as chromosomal DNA
- Neutralisation (K acetate) results in aggregation of chromosomal DNA, protein-SDS complexes and high molecular weight RNA
- The precipitate is removed by centrifugation and the plasmid DNA remains in solution
- Plasmid DNA is purified using silica or +ve charged ion matrix in a column
- Plasmid DNA binds to the matrix at high set concentration and contaminants can be washed away
- Pure plasmid DNA is eluted from the matrix under low salt conditions and collected
How do we check plasmid DNA?
Agarose gel electrophoresis
Why does agarose gel electrophoresis work? (3)
- DNA is negatively charged
- DNA fragments separated on basis of their size by applying an electric current
- Allows DNA fragments to be detected using UV light
What are the steps in agarose gel electrophoresis? (6)
- Add samples into wells
- Electric current passed through the gel
- DNA fragments move according to size, smallest the furthest
- Compared to a size marker to identify
- plot graph
- Graph can be used to determine the length of unknown fragments based on distance
What is DNA blotting?
- DNA separated through an agarose gel and transferred onto a membrane (denatured)
- A radioactively labelled single stranded DNA probe is incubated with membrane and will hybridise with corresponding DNA (pairing)
- After washing to eliminate non-specific binding, the membrane is exposed to an x-ray film (fiend specific DNA)
- After the film is developed the band on the film shows the corresponding DNA
What is cDNA? (3)
- Double stranded DNA made by copying eukaryotic mRNA
- mRNA is copied into single stranded DNA using reverse transcriptase enzyme
- This single stranded DNA is made double stranded using a standard DNA polymerase or a great stable DNA polymerase
Why is cDNA useful? (3)
- It is a DNA copy of a gene which does not contain the introns which are usually present in copies
- cDNA can be used to make the protein product of the gene in either pro/eukaroytoic system
- Bacteria lack introns and the machinery to remove them, but using cDNA means this is not a problem
How is cDNA constructed? (5)
- 3’ to 5’ + viral reverse transcriptase
- Hairpin loop occurs on cDNA + NaOH (removes RNA)
- Hairpin extends + DNA polymerase
- cDNA extends fully + S1 nuclease (snap the hairpin)
- double stranded DNA
