Lecture 6: DNA Transduction into Cells Flashcards
DNA is added into cells for a number of reasons:
It can be used to replicate or amplify if for further uses
It can also be used to induce the expression of particular proteins within the cell
DNA is also added to modify the genome of the cell
DNA can be aded to cells in different ways (mention the ways):
How does the process vary and what does it depend on?
DNA can be added to cells in different ways with this process varying depending on the cell type.
Transformation | is used for Bacterial and yeast cells
Transfection | is used for eukaryotic cells
Electroporation | can be used for all cell types
Transformation
Chemically competent bacteria are grown rapidly into their log phase before they are spun down in a centrifuge and washed with divalent cations (e.g. calcium) within salts
Yeast cells are transformed using lithium acetate in combination with PEG (polyethylene glycol).
Transfection
Eukaryotic cells are incubated with lipid or calcium phosphate along with the DNA
The result is DNA is fused with the plasma membrane with the DNA moved into the cell
The lipid or calcium phosphate allow for fusion for endocytosis of the two compounds respectively and this allows the DNA access into the cell
Electroporation
Electroporation can be used for all cell types.
With this technique, a high voltage is passed across the cell for a very short amount of time as this temporarily open the plasma membrane and allows the DNA to be taken up by the cells.
The DNA obtained through the use of reverse transcriptase, specific primers and amplification by PCR is maintained by…
…cloning it into bacteria (can be stored in freezers for long periods of time).
The PCR fragment needs to be ligated into a plasmid vector to do this.
The specific genes should contain…
blunt ends meaning the plasmid vector should also contain blunt ends when it is cut
- The PCR is then combined with the plasmid with DNA ligase then sticky the ends together to form the vector.
- The vector is then transformed into competent bacteria with this bacteria plated on selective media incorporating blue/white selection (see below).
The vector will contain:
A gene that corresponds to the selective media (e.g. ampicillin resistant vector will be grown in an agar plate containing ampicillin)
Must also contain an origin of replication that allows the vector to be amplified within the bacterial cell
There is also a multiple cloning site which is where restriction enzymes will cleave to allow insertion of the target gene
If the gene is to be expressed within the target cell, RNA binding sites within the vector are also required
An RNA polymerase binding site is also usually included within the target gene
What can affect the process?
The DNA Polymerase used.
Taq polymerase for example leaves a single, overhanging A at the 3’ end of the PCR product. Meanwhile the vector has overhanging Ts at both it ends.
This not only make the insertion of the PCR product seamless (the A and T are attracted through hydrogen bonding initially with DNA ligase completing the job of sealing the nicks), it also prevents re-circularisation
The site of insertion of the new gene may also be within a _____ gene
The site of insertion of the new gene may also be within a LacZ gene
If unwanted DNA is inserted this inactivates the β-galactosidase and this can be tested for on the agar plates
A substrate for β-galactosidase called Xgal turns blue when acted on by its enzyme so this substrate is used in the plate (the blue/white section)
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Bacterial colonies will turn white if the correct DNA has been inserted
After Insertion…
*
…a single colony is picked and this is grown in the presence of antibiotic
Some of the bacterial is taken with a little retained to maintain the original sample
DNA is extracted from the culture and this is checked that it is the specific DNA we need