Purification of plasmid DNA

Question 1

What are plasmids?

Answer 1

Self-replicating circles of double stranded DNA found in many bacterial cells.

Question 2

Why is purification of plasmid more difficult than purification of total bacterial DNA?

Answer 2

The plasmid DNA must be separated from the bacterial chromosomal DNA which is more difficult.

Question 3

What are some physical differences between plasmid DNA and bacterial DNA?

Answer 3

Plasmid DNA is much smaller than bacterial DNA and plasmid DNA remains circular instead of forming linear fragments during preparation of the cell extract when broken down.

Question 4

What is one way to separate the plasmid DNA from the bacterial DNA?

Answer 4

The cells can be lysed in an isotonic solution (so minimal damage to chromosomal DNA occurs) and a detergent (Triton X-100) can be used to rupture the membrane. The chromosomal DNA remains in large pieces and co-pellets with the cell debris upon centrifugation, whereas the plasmids remain in solution which can be pipetted to a fresh tube.

Question 5

What steps can be taken to further purify the plasmid DNA?

Answer 5

Ehtidium bromide-caesium chloride density gradient centrifugation can be used to remove residual chromosomal DNA is ultra pure plasmid DNA is needed.

Question 6

How does ethidium bromide-caesium chloride work to purify DNA?

Answer 6

Ethidium bromide intercalates between the strands of the double helix to reduce the buoyant density. The density of linear DNA is reduced more than supercoiled DNA (undamaged plasmids) which can allow the two to be separated on a caesium-bromide gradient.

Question 7

What happens after ethidium bromide has been added to the solution?

Answer 7

The solution is mixed with caesium chloride and is centrifuged at a high speed for at least 24 hours. A density gradient is produced Different bands will be formed in the gradient which will depend on the buoyant density.

Question 8

Where will the different components in the ethidium bromide-caesium chloride density centrifugation form?

Answer 8

DNA will migrate to around 1.7g cm-3 (middle of tube), protein will rise to the top of the tube (lower buoyant density) and RNA will form pellets at the bottom.

Question 9

How can the plasmid DNA be removed from the chromosomal DNA?

Answer 9

As the linear and supercoiled DNA will band separately (linear above the hypercoiled) the plasmid DNA can be removed with a hypodermic syringe.

Question 10

How can contaminant EtBr and CsCl be removed from the plasmid sample after centrifugation?

Answer 10

EtBr can be removed with butanol and CsCl can be removed by dialysis.

Question 11

What is a useful tag on mRNA that allows it to be separated from the rest of RNA?

Answer 11

mRNA is polyadenylated at the 3’ end (string of adenines attached) which acts as a useful tag to select the mRNA from the rest of the RNA.

Question 12

What is the method used to separate the mRNA?

Answer 12

Oligo(dT) chromatography.

Question 13

What is the first step in the basic method for Oligo(dT) chromatography?

Answer 13

Firstly, prepare a small column containing Oligo(dT) bound to a cellulose matrix.

Question 14

What happens in the second stage of Oligo(dT) chromatography?

Answer 14

The total RNA is added in a high salt buffer and the Poly(A) tails of the mRNA will bind to the oligo-dT by hydrogen bonding.

Question 15

What is the third step in oligo(dT) chromatography?

Answer 15

A medium salt buffer can be added that washes of rRNA and tRNA - these are not bound to the oligo-dT.

Question 16

What is added after the third step in oligo-dT chromatography?

Answer 16

A buffer without salt is then added to the column to wash off the polyA RNA that has been stuck to the oligo-dt.

Question 17

How can the RNA then be concentrated in the final step of oligo-dT chromatography?

Answer 17

Ethanol precipitation can be used to concentrate the RNA.

Question 18

What is a variation of oligo-dT chromatography?

Answer 18

The oligo-dT can be attached to magnetic beads and a magnet can be used to isolate the mRNA, as everything else is washed off. The mRNA will be stuck to the magnet.

Question 19

How does purification of total eukaryotic DNA differ from the purification of DNA from bacteria?

Answer 19

A convenient material will be chosen such as young leaves, liver cells, the material is usually mechanically disrupted such as grinding it in liquid nitrogen or sonication and extra steps may be required and materials may be challenging and require special procedures.

Question 20

What are some examples of enzymes that are used to manipulate nucleic acids?

Answer 20

Nucleases, ligases, polymerases and modifying enzymes.

Question 21

What are the functions of each of the different types of enzymes used to manipulate nucleic acids?

Answer 21

Nucleases cut, shorten or degrade nucleic acids, ligases join molecules together, polymerases make copies of nucleic acid molecules and mofiying enzymes remove or add chemical groups.

Question 22

What are the two subclasses of nucleases?

Answer 22

Exonucleaes that remove nucleotides one at a time from the ends of a nucleic acid molecule and endonucleases that are able to break internal phosphodiester bonds within a nucleic acid molecule.

Question 23

Give two examples of one type of nuclease.

Answer 23

Bal31 is an exonuclease that digests the ends of DNA and can be used to shorten DNA molecules. The degree of digestion is dependent on the time and concentration. Exonuclease III removes 3’ termini of DNA molecules and leaves single stranded ends or whole molecules, dependent on the conditions of the reaction.

Question 24

Give two examples of the other type of nuclease.

Answer 24

S1 is an endonuclease that digests only single-stranded DNA, leaving double stranded DNA in tact. It can be used to remove single stranded ends prior to cloning or to differentiate between single and double stranded DNA. Deoxyribonuclease I (DNase I) chops up all DNA and is useful to get rid of DNA or to produce small fragments.

Question 25

What are restriction endonucleases?

Answer 25

A class of endonucleases with many members that recognise and cut DNA at specific sequences, usually 4 or 6 base pairs long. The nature of the cut is specific to the enzyme.

Question 26

What other kinds of nucleases are there?

Answer 26

Nucleases that degrade RNA such as ribonuclease (RNase). This degrades single or double stranded RNA and can be used to remove RNA that contaminates DNA solutions. It is a robust enzyme that does not become denatured under boiling. It is hard to destroy and an RNase-free environment must be maintained if you are working with RNA.

Question 27

What is the natural function of ligases?

Answer 27

Repair single stranded breaks in DNA.

Question 28

What use do ligases have in cloning?

Answer 28

Joining DNA molecules together for cloning.

Question 29

What do polymerases do?

Answer 29

Make copies of nucleic acids that are complementary to a template.

Question 30

What 3 components do polymerases require?

Answer 30

A single stranded template, short primer sequences that are base paired to the template (a double stranded region for initiation is created) and a supply of nucleotides.

Question 31

Give an example of a polymerase.

Answer 31

DNA polymerase I from e.coli that repairs nicks in DNA by synthesising a new strand. It has a proof-reading capability as it replaces any existing strand as it proceeds, therefore having a dual activity of DNA polymerisation and DNA degradation

Question 32

What is the Klenow fragment of DNA polymerase I?

Answer 32

A part of the polymerase that has the proof reading capability removed (323 amino acids). It will only synthesise the complementary strand. This is used in DNA sequencing.

Question 33

What is reverse transcriptase?

Answer 33

It is from retroviruses and makes DNA copies of their RNA genomes. Its method of action resembles the Klenow fragment of DNA pol I, using RNA instead of DNA.

Question 34

What can reverse transcriptase be used for?

Answer 34

It can be used to synthesise complementary DNA sequences of RNA. It can be used for cDNA cloning - cloning mRNA.