Lecture 6: DNA Transduction into Cells Flashcards

1
Q

DNA is added into cells for a number of reasons:

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

DNA can be aded to cells in different ways (mention the ways):

How does the process vary and what does it depend on?

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

Transformation

A

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).

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

Transfection

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

Electroporation​

A

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.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

The DNA obtained through the use of reverse transcriptase, specific primers and amplification by PCR is maintained by…

A

…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.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

The specific genes should contain…

A

blunt ends meaning the plasmid vector should also contain blunt ends when it is cut

  1. The PCR is then combined with the plasmid with DNA ligase then sticky the ends together to form the vector.
  2. The vector is then transformed into competent bacteria with this bacteria plated on selective media incorporating blue/white selection (see below).
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

The vector will contain:

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

What can affect the process?

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

The site of insertion of the new gene may also be within a _____ gene

A

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)

|

Bacterial colonies will turn white if the correct DNA has been inserted

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

After Insertion…

*

A

…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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly