lecture 9 Practical applications of molecular biology Flashcards
what is DNA cloning?
*Involves isolating and creating identical copies of a specific gene – maybe to test its function.
- Start with the DNA sequence of the gene of interest
- Design PCR primers to clone the gene
*This will involve knowing the destination of the PCR product – the expression vector - Ligation
*Introduce the PCR product into the plasmid in the correct place and orientation
4.Transformation of competent cells with plasmid
*Using calcium chloride method
5.Selection of bacterial clones
*Analysis and troubleshooting
How do we get multiple, identical copies of the gene we’re interested in?
PCR!
Question: what will we do with the PCR product when we have it?
Answer: we need to clone it
DNA cloning involves:
1.Creating identical copies of a specific gene with PCR
2.Using restriction nucleases to cut the desired gene
3.Ligating it into a plasmid, and inserting it into bacteria like E. coli
Elements of an expression plasmid
*Origin of replication – allows the plasmid to replicate inside the bacterium
*Antibiotic resistance gene – confers resistance to the bacteria containing the plasmid – allows you to select only for the bacteria that you have successfully introduced the plasmid into
*Selectable marker – maybe another antibiotic resistance gene or something else that allows for selection of the plasmid
*Promoter – chosen to produce high expression of the inserted gene – often preferable to using the promoter that was part of the cloned gene
*5’ and 3’ primer sites – defined by the manufacturer as the primers you can use to confirm the size of the inserted gene – no inserted gene = v. small product
*Restriction sites – specific DNA sequences cut by specific restriction nucleases
PCR conditions
Optimum annealing temperature is required to ensure specific binding of primers.
(a)Too high –> no PCR product
(b)Too low –> too many non-specific PCR products
(c)Optimum temp –> desired PCR product
*Optimal melting temperatures (Tm) for primers range between 52-58°C, although the range can be expanded to 45-65°C. The final Tm for both primers should differ by no more than 5°C.
*The 3’ end of primers should contain a G or C in order to clamp the primer and prevent “breathing” of ends, increasing priming efficiency.
*The 3’ ends of a primer set, should not be complementary to each other, nor can the 3’ end of a single primer be complementary to other sequences in the primer. These two scenarios result in formation of primer dimers and hairpin loop structures, respectively
*Di-nucleotide repeats (e.g., GCGCGCGCGC or ATATATATAT) or single base runs (e.g., AAAAA or CCCCC) should be avoided as they can cause slipping along the primed segment of DNA and or hairpin loop structures to form. If unavoidable due to nature of the DNA template, then only include repeats or single base runs with a maximum of 4 bases
cutting our plasmid and PCR product with EcoRI and HindI
*Commonly into E. coli DH5-alpha cells
*Engineered by American biologist Douglas Hanahan to maximise transformationefficiency.
*The cells are competent and often used with calcium chloride transformation to insert the desired plasmid.
*Competence is the ability of a cell to alter its genetics by taking up extracellular (“naked”) DNA from its environment in the process called transformation.
*DH5-alpha cells are treated to make them transiently permeable to DNA.
Calcium chloride transformation
*The addition of calcium chloride to a cell suspension promotes the binding of plasmid DNA to lipopolysaccharides (LPS).
*Positively charged calcium ions attract both the negatively charged DNA backbone and the negatively charged groups in the LPS inner core.
*The plasmid DNA can then pass into the cell upon heat shock, where chilled cells (4oC) are heated to a higher temperature (42oC) for a short time.
But what if there’s no haemolysis?
*Maybe yaoF is not hemolysin at all
*Maybe it is but it’s non-functional in E. coli
*Maybe the cloning failed?
-The ligation reaction contained mostly uncut backbone plasmid?
*The PCR failed to produce the correct gene product
