Purification of nucleic acids 1

Question 1

What is gene cloning?

Answer 1

Making multiple copies of a gene of interest. It involves the purification and amplification of nucleic acid molecules in a cloning vector or via PCR.

Question 2

What is the first step in basic cloning?

Answer 2

Inserting the DNA fragment into a vector to form a recombinant molecule (containing DNA from two sources).

Question 3

What are the following steps in basic DNA cloning?

Answer 3

The molecule is transported into a bacterial cell and then replicated. Bacterial colonies are produced that contain the recombinant molecule and each colony is a clone of the identical host cells, containing the recombinant molecule.

Question 4

What components are required for PCR?

Answer 4

A thermostable DNA polymerase (Taq), short DNA primers, a supply of nucleotides and a thermocycler.

Question 5

Why would gene cloning be used if PCR could be used instead?

Answer 5

Gene cloning can provide a pure sample of the gene of interest and allows the genes to be modified and introduced into the organism from which it was isolated from and other organisms, even if they are in other kingdoms. It also allows genes and genomes to be sequenced, and has many uses in medicine, diagnostics, forensics and biotechnology. PCR is more selective as the sequence must be known beforehand.

Question 6

What are some issues with PCR?

Answer 6

Sequence information must be known to design the primers, Taq polymerase has an error rate and there is a limitation of the size of the base pairs that can be used (5000 - this is shorter than many genes).

Question 7

Why is the production of animal protein useful?

Answer 7

It can be used to produce hormones such as human growth factor.

Question 8

What are the different types of DNA?

Answer 8

Chromosomal, mitochondrial, plastid, plasmid and viral.

Question 9

What are the different types of RNA?

Answer 9

mRNA, rRNA, tRNA and viral RNA.

Question 10

What is the first essential step in nucleic acid purification?

Answer 10

Disruption of the material such as lysis, grinding or sonication.

Question 11

What is the second step in nucleic acid purification?

Answer 11

Removal of the protein such as using phenol extraction, which denatures the protein or the digestion of the protein using an enzyme such as proteinase K.

Question 12

What is the third step in nucleic acid purification?

Answer 12

Using ethidium bromide-caesium chloride density gradient centrifugation or column chromatography to purify the nucleic acid.

Question 13

What is the final step in nucleic acid purification?

Answer 13

The concentration of the nucleic acid by precipitation using ethanol and salt.

Question 14

What are the two types of media that can be used to grow a bacterial culture?

Answer 14

A defined medium that has known chemical components or an undefined medium that contains a mixture of organic extracts.

Question 15

How can cell number be estimated when growing bacteria?

Answer 15

Using optical density that can be measured by a spectrophotometer. As cells increase in number the medium becomes more cloudy and less light can get through.

Question 16

When should cells be harvested when growing the bacterial culture?

Answer 16

At the late exponential phase.

Question 17

How can cells be harvested when they have been left to the late exponential phase?

Answer 17

Using centrifugation. The cells will form a pellet at the bottom of the tube and the supernatant (excess liquid) can be discarded.

Question 18

What happens after the cells have been harvested?

Answer 18

The bacterial pellet can be suspended in a lysis solution containing enzymes and detergents that will disrupt the cell wall and membrane. THe cell extract can then be centrifuged to remove cell debris, leaving the nucleic acids and protein behind in solution.

Question 19

What must be removed from the bacterial cell extract?

Answer 19

Proteins and RNA.

Question 20

How can the unwanted substances be removed from the bacterial cell extract?

Answer 20

A mixture of phenol-chloroform and proteinase can be used to degrade the contaminants and then use ion-exchange chromatography to separate the DNA from the contaminants.

Question 21

What is the role of each of the components involved in removing unwanted substances from the bacterial cell extract?

Answer 21

Phenol denatures the proteins to form a white fluffy layer and chloroform helps to reduce interphase (the fuzzy border between layers).

Question 22

What is the role of centrifugation in the degradation of contaminants?

Answer 22

It separates the aqueous layer (DNA and RNA) from the organic layer

Question 23

How does ion exchange work?

Answer 23

As DNA and RNA are negatively charged, they will bind to a positively charged resin. This separates molecules according to how tightly they bind to electrically charged particles present in a chromatographic matrix or resin. Increasing the salt concentration elutes (washes off) bound components. DNA elutes at high salt concentrations as it is tightly bound.

Question 24

Why does the DNA need to be concentrated?

Answer 24

As downstream application will often require high concentrations of DNA.

Question 25

What is the method used to concentrate DNA?

Answer 25

Ethanol precipitation. This is when ethanol is added with salt to precipitate the DNA, followed by the mixture being cooled for 1-24 hours. The DNA can be collected with a glass rod if in high concentrations or centrifuged if in low concentrations.

Question 26

What extra purification steps may need to be taken?

Answer 26

Ethidium bromide-caesium chloride density gradient centrifugation.

Question 27

What is the ratio for a measure of purity?

Answer 27

A260/A280.

Question 28

What is the ratio for pure DNA?

Answer 28

1.8

Question 29

What reduces the ratio when measuring purity?

Answer 29

Contamination with protein or phenol.